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Sample records for deoxynucleotidyltransferase-mediated dutp nick

  1. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using bench top flow cytometer for evaluation of sperm DNA fragmentation in fertility laboratories: protocol, reference values, and quality control.

    Science.gov (United States)

    Sharma, Rakesh; Ahmad, Gulfam; Esteves, Sandro C; Agarwal, Ashok

    2016-02-01

    The purpose of this study is to provide a detailed protocol and quality control steps for measuring sperm DNA fragmentation (SDF) by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay using a new bench top flow cytometer, determine the reference value of SDF, and assess sensitivity, specificity, and distribution of SDF in infertile men and controls with proven and unproven fertility. Semen specimens from 95 controls and 261 infertile men referred to a male infertility testing laboratory were tested for SDF by TUNEL assay using Apo-Direct kit and a bench top flow cytometer. Percentage of cells positive for TUNEL was calculated. Inter- and intraobserver variability was examined. TUNEL cutoff value, sensitivity, specificity, and distribution of different cutoff values in controls and infertile patients were calculated. The reference value of SDF by TUNEL assay was 16.8 % with a specificity of 91.6 % and sensitivity of 32.6 %. The positive and negative predictive values were 91.4 and 33.1 %, respectively. The upper limit of DNA damage in infertile men was significantly higher (68.9 %) than that in the controls (19.6 %). TUNEL assay using flow cytometry is a reproducible and easy method to determine SDF. At a cutoff point of 16.8 %, the test showed high specificity and positive predictive value. The results of this test could identify infertile men whose sperm DNA fragmentation does not contribute to their infertility and confirm that a man who tests positive is likely to be infertile due to elevated sperm DNA fragmentation.

  2. 精子染色质扩散试验及末端转移酶标记法检测精子脱氧核糖核酸完整性%Measurement of sperm DNA integrity by sperm chromatin dispersion test and TdT-mediated dUTP nick end labeling assay

    Institute of Scientific and Technical Information of China (English)

    张丽红; 王磊光; 邱毅; 王克华; 李娟; 张梅心; 刘静; 贾颐舫; 吴爱华; 张爱东

    2009-01-01

    目的 比较精子染色质扩散试验(SCD)和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测精子DNA完整性.方法 对20名已生育的成年健康男性和32例不育患者的精液用上游法筛选得到活动精子,同时进行SCD和TUNEL.结果 不育组和生育组DNA损伤精子百分率的SCD检测结果分别为(12.8±5.8)%和(7.6±3.3)%,2组比较差异有统计学意义(t=3.576,P=0.001);TUNEL检测结果分别为(11.1±5.1)%和(6.8±2.8)%,2组比较差异有统计学意义(t=3.467,P=0.001).SCD与TUNEL检测结果在不育组(r=0.841,P=0.000)和生育组(r=0.823,P=0.000)均具有明显的线性相关关系.生育组SCD检测结果与TUNEL检测结果比较差异无统计学意义(t=1.996,P=0.060),而不育组SCD检测结果明显高于TUNEL检测结果,差异有统计学意义(t=3.023,P=0.005).结论 精子DNA损伤与男性不育有关.与TUNEL比较,SCD是一种更为准确、简单、廉价的检测精子DNA完整性的方法.%Objective To compare the effects of sperm chmmatin dispersion(SCD)test and TDTmediated dUTP nick end labeling(TUNEL)assay in assessing the DNA fragmentation in human spem.Methods Motile sperms were isolated from the semen samples obtained from 20 healthy fertile men and 32clinically infertile pailents by swim-up technique,and underwent SCD and TUNEL to analyze the DNA fragmentation.Results The rate of sperm with DNA damage of the infertile pailents was 12.8%±5.8%tested by SCD,significantly higher than that of the healtby fertile men(7.6%±3.3%,t=3.576,P=0.001),and the rate of sperm with DNA damage of the infertih patients wag 11.1%±5.1%tested by TUNEL assay.significantly higher than that of the healthy fertile men(6.8%±2.8%.t=3.467,P=0.001).The proportion of sperm cell wlth abnormal DNA integrity meagured by SCD test wag correlated strongly with that determined by TUNEL for the infertile men(r=0.841.P=0.000)and for the fertile men too(r=0.823.P=0.000).The rote of sperm with DNA damage

  3. Simple method for preparation of fluor/hapten-labeled dUTP.

    Science.gov (United States)

    Nimmakayalu, M; Henegariu, O; Ward, D C; Bray-Ward, P

    2000-03-01

    Many projects, such as multiplex-fluorescence in situ hybridization (M-FISH) karyotyping, require the use of relatively large amounts of multiple fluor- or hapten-labeled nucleotides for the preparation of DNA probes. Such a requirement makes these experimental approaches prohibitively expensive for many researchers. The cost of such nucleotides can be reduced approximately 99% by purchasing the chemical precursors, fluor or hapten succinimidyl esters and 5-(3-aminoallyl)-2'-deoxyuridine 5' triphosphate (AA-dUTP), and performing the simple coupling/purification described here. It is possible to finish four to ten different fluor/hapten dUTP preparations of 2.5 microM scale within a 24 h period. The reagent cost for each preparation ranges from $33-$237 per microM, depending on the fluor/hapten. This laboratory uses such nucleotide preparations to prepare FISH probes by nick translation or PCR amplification.

  4. Modeling the Forced Extension of Nicked DNA

    Science.gov (United States)

    Balaeff, Alexander; Craig, Stephen; Beratan, David

    2007-03-01

    The design and study of DNA-based nanodevices has been a topic of considerable interest in the last decade. While the applications of classical continuous DNA structures have been thoroughly studied, nicked DNA structures, i.e., ones that contains breaks (``nicks'') in one or both DNA backbone chains, have received much less attention. Recently, Kersey et al. (JACS, 2004) reported the force spectroscopy of long DNA chains with periodic nicks, self-assembled from short DNA oligomers. We attempt to model the experimental force-extension profiles in a series of steered molecular dynamics simulations. The simulated all-atom model of a basic unit of the long self-assembled chain, a 16bp-long DNA segment with a nick in the middle of one strand, is extended by applying either a constant force or a moving harmonic potential to the DNA ends. The computed force-extension profiles are compared to those for a non-nicked DNA; the dynamics of structural changes in the nicked DNA during the forced extension is discussed. A theoretical framework is established to link the extension and rupture in the simulated basic unit to the corresponding events in the long self-assembled chain.

  5. Roman for unge - og deres voksne - fra Nick Hornby

    DEFF Research Database (Denmark)

    Haarder, Jon Helt

    2007-01-01

    Nick Hornby: SLAM. Oversat fra engelsk. af Jan Hansen. 280 sider, 249 kr. Lindhardt og Ringhof. Fire stjerner Udgivelsesdato: 071207......Nick Hornby: SLAM. Oversat fra engelsk. af Jan Hansen. 280 sider, 249 kr. Lindhardt og Ringhof. Fire stjerner Udgivelsesdato: 071207...

  6. GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism.

    Science.gov (United States)

    Chan, Ying Wai; West, Stephen

    2015-12-15

    Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells.

    Science.gov (United States)

    Williams, Drew; Norman, Grant; Khoury, Chamel; Metcalfe, Naomi; Briard, Jennie; Laporte, Aimee; Sheibani, Sara; Portt, Liam; Mandato, Craig A; Greenwood, Michael T

    2011-02-01

    The identification of novel anti-apoptotic sequences has lead to new insights into the mechanisms involved in regulating different forms of programmed cell death. For example, the anti-apoptotic function of free radical scavenging proteins supports the pro-apoptotic function of Reactive Oxygen Species (ROS). Using yeast as a model of eukaryotic mitochondrial apoptosis, we show that a cDNA corresponding to the mitochondrial variant of the human DUT gene (DUT-M) encoding the deoxyuridine triphosphatase (dUTPase) enzyme can prevent apoptosis in yeast in response to internal (Bax expression) and to exogenous (H(2)O(2) and cadmium) stresses. Of interest, cell death was not prevented under culture conditions modeling chronological aging, suggesting that DUT-M only protects dividing cells. The anti-apoptotic function of DUT-M was confirmed by demonstrating that an increase in dUTPase protein levels is sufficient to confer increased resistance to H(2)O(2) in cultured C2C12 mouse skeletal myoblasts. Given that the function of dUTPase is to decrease the levels of dUTP, our results strongly support an emerging role for dUTP as a pro-apoptotic second messenger in the same vein as ROS and ceramide.

  8. Essays on the future in honor of Nick Metropolis

    CERN Document Server

    Rota, Gian-Carlo

    2000-01-01

    This collection represents a unique undertaking in scientific publishing to honor Nick Metropolis. Nick was the last survivor of the World War II Manhattan Project in Los Alamos, and was an important member of the Los Alamos national Laboratory until his death in October, 1999. In this volume, some of the leading scientists and humanists of our time have contributed essays related to their respective disciplines, exploring various aspects of future developments in science and society, philosophy, national security, nuclear power, pure and applied mathematics, physics and biology, particle physics, computing, and information science.

  9. March 2011 DMM Podcast: an interview with Nick Hastie

    Science.gov (United States)

    2012-01-01

    SUMMARY Excerpts from an interview with Nick Hastie, Director of the MRC Human Genetics Centre in Edinburgh, discussing his early career, his involvement in large-scale genetic studies and his exciting new findings on WT1. Narrated by Sarah E. Allan. To listen to this podcast, visit http://www.biologists.com/DMM/podcasts/index.html.

  10. Oxygen and Metastasis: A Conversation with Dr. Nick Restifo

    Science.gov (United States)

    Dr. Nick Restifo, a senior investigator in NCI’s Center for Cancer Research, discusses his recently published study finding that Oxygen, a molecule necessary for life, paradoxically aids cancer metastasis to the lung by impairing cancer-killing immune cells.

  11. Nick Park pani Wallace'ile ja Gromitile libaküüliku needuse / Mart Rummo

    Index Scriptorium Estoniae

    Rummo, Mart

    2005-01-01

    Uus animafilm "Wallace ja Gromit : Libaküüliku needus" ("Wallace & Gromit : The Curse of the Were-Rabbit") : režissöör Nick Park : Suurbritannia 2005. Selle loojast ja tema loometeest. Lisatud : Nick Park (filmograafia, auhindu), "Teised Nick Parkist" (Lauri Kaare, Mait Laas, Riho Unt)

  12. Nick Park pani Wallace'ile ja Gromitile libaküüliku needuse / Mart Rummo

    Index Scriptorium Estoniae

    Rummo, Mart

    2005-01-01

    Uus animafilm "Wallace ja Gromit : Libaküüliku needus" ("Wallace & Gromit : The Curse of the Were-Rabbit") : režissöör Nick Park : Suurbritannia 2005. Selle loojast ja tema loometeest. Lisatud : Nick Park (filmograafia, auhindu), "Teised Nick Parkist" (Lauri Kaare, Mait Laas, Riho Unt)

  13. Studying repair of a single protein-bound nick in vivo using the Flp-nick system

    DEFF Research Database (Denmark)

    2012-01-01

    damage mimics a stabilized topoisomerase I-DNA cleavage complex. DNA topoisomerases are ubiquitous enzymes that relieve topological stress in the DNA arising during DNA replication or transcription. During this process, they make transient enzyme-DNA cleavage complexes, which normally are reversed...... by a rapid ligation step. However, aberrant long-lived enzyme-DNA complexes may occur frequently due to either endogenous or exogenous damage, and the cellular repair machinery therefore needs to be able to eliminate this type of damage. This chapter describes how to establish the Flp-nick system in S....... cerevisiae, how to detect FlpH305L at the FRT site using a modified chromatin immunoprecipitation assay where formaldehyde fixation is omitted, and how to monitor nicking at the FRT site by alkaline denaturing gel analysis...

  14. Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase.

    Science.gov (United States)

    Téllez-Sanz, Ramiro; Yassin, Zeyad; Bernier-Villamor, Victor; Ortiz-Salmerón, Emilia; Musso-Buendia, Juan Alexander; Barón, Carmen; Ruíz-Pérez, Luis Miguel; González-Pacanowska, Dolores; García-Fuentes, Luis

    2007-08-01

    dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.

  15. Structural Basis for Nick Recognition by a Minimal Pluripotent DNA Ligase

    Energy Technology Data Exchange (ETDEWEB)

    Nair,P.; Nandakumar, J.; Smith, P.; Odell, M.; Lima, C.; Shuman, S.

    2007-01-01

    Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-{angstrom} crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO{sub 4} nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a {beta}-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO{sub 4} termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.

  16. Aortic valve replacement with the Nicks annulus enlargement procedure 12 years after mitral valve replacement.

    Science.gov (United States)

    Kazama, S; Kurata, A; Yamashita, Y

    1999-10-01

    An aortic valve replacement was successfully performed employing the Nicks annulus enlargement procedure in a case of aortic valve stenosis with small annulus 12 years after mitral valve replacement. Previous mitral valve replacement does not preclude feasibility of the Nicks procedure.

  17. DNA fragments assembly based on nicking enzyme system.

    Directory of Open Access Journals (Sweden)

    Rui-Yan Wang

    Full Text Available A couple of DNA ligation-independent cloning (LIC methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC and Uracil-Specific Excision Reagent (USER was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB.

  18. NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Shoji, Wataru [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Suenaga, Yusuke, E-mail: ysuenaga@chiba-cc.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Kaneko, Yoshiki; Islam, S.M. Rafiqul; Alagu, Jennifer [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Yokoi, Sana [Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Nio, Masaki [Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Nakagawara, Akira, E-mail: nakagawara-a@koseikan.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan)

    2015-06-05

    NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas. - Highlights: • NCYM promotes cleavages of MYC and MYCN to produce Myc-nick in vitro. • NCYM increases Myc-nick production in MYCN-amplified neuroblastoma cells. • NCYM knockdown decreases Myc-nick production and induces apoptosis at G2/M phase.

  19. Two DNA-binding and Nick Recognition Modules in Human DNA Ligase III*

    OpenAIRE

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Tomkinson, Alan E.; Ellenberger, Tom

    2008-01-01

    Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bin...

  20. Pangahävitaja Leeson : "Ma kahetsen" / Nick Leeson ; interv. Argo Ideon

    Index Scriptorium Estoniae

    Leeson, Nick

    2007-01-01

    Briti börsimaakler Nick Leeson, kes pankrotistas Baringsi panga ning hävitas 17 miljardit krooni, selgitab, miks ta ei suutnud oma tegevust lõpetada ka siis kui kahjud olid kasvanud juba hiigelsuureks

  1. Pangahävitaja Leeson : "Ma kahetsen" / Nick Leeson ; interv. Argo Ideon

    Index Scriptorium Estoniae

    Leeson, Nick

    2007-01-01

    Briti börsimaakler Nick Leeson, kes pankrotistas Baringsi panga ning hävitas 17 miljardit krooni, selgitab, miks ta ei suutnud oma tegevust lõpetada ka siis kui kahjud olid kasvanud juba hiigelsuureks

  2. Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis.

    Science.gov (United States)

    Curry, John D; Geier, Jamie K; Schlissel, Mark S

    2005-12-01

    Variable (diversity) joining (V(D)J) recombination is initiated by the introduction of single-strand DNA breaks (nicks) at recombination signal sequences (RSSs). The importance and fate of these RSS nicks for the regulation of the V(D)J rearrangement and their potential contribution to genomic instability are poorly understood. Using two new methodologies, we were able to detect and quantify specific RSS nicks introduced into genomic DNA by incubation with recombination-activating gene proteins in vitro. In vivo, however, we found that nicks mediated by recombination-activating gene (RAG) proteins were detectable only in gene segments associated with RSSs containing 12-base pair spacers but not in those containing 23-base pair spacers. These data support a model of capture rather than synapsis for pairwise RSS cleavage during V(D)J recombination.

  3. Nick Ransford: 'There is a job to do so let's get on with it'.

    Science.gov (United States)

    Ransford, Nick; Doherty, Ruth

    2014-03-01

    Nick Ransford is a consultant in special care dentistry at Birmingham Community Healthcare NHS Trust. He has over 25 years' experience working with adults with disabilities and medical conditions across the spectrum.

  4. An Analysis on the Maturity of Nick in The Great Gatsby

    Institute of Scientific and Technical Information of China (English)

    刘莎莎

    2014-01-01

    Nick Carraway is one of the main characters as well as the narrator in F. Scott Fitzgerald’s The Great Gatsby. This pa-per applies the theory of Bildungsroman and analyzes the changes of Nick in characters, conscience as well as responsibilities from the following aspects:guides of maturity and epiphany during maturity, and reveals his ultimate maturity both physically and men-tally after epiphany, thus shows the changes and maturity of him.

  5. The gastroprotective effect of pogostone from Pogostemonis Herba against indomethacin-induced gastric ulcer in rats.

    Science.gov (United States)

    Chen, Xiao-Ying; Chen, Hai-Ming; Liu, Yu-Hong; Zhang, Zhen-Biao; Zheng, Yi-Feng; Su, Zu-Qing; Zhang, Xie; Xie, Jian-Hui; Liang, Yong-Zhuo; Fu, Lu-Di; Lai, Xiao-Ping; Su, Zi-Ren; Huang, Xiao-Qi

    2016-01-01

    Pogostemonis Herba, known as "Guang-Huo-Xiang" in Chinese, has been widely used in the treatment of gastrointestinal dysfunction. Pogostone is one of the major constituents of Pogostemonis Herba. The aim was to scientifically evaluate the possible gastroprotective effect and the underlying mechanisms of pogostone against indomethacin-induced gastric ulcer in rats. Rats were orally treated with vehicle, lansoprazole (30 mg/kg) or pogostone (10, 20 and 40 mg/kg) and subsequently exposed to acute gastric lesions induced by indomethacin. Gross evaluation, histological observation, gastric mucosal superoxide dismutase activity, glutathione content, catalase activity, malonaldehyde level and prostaglandin E2 production were performed. Immunohistochemistry and reverse transcription polymerase chain reaction for cyclooxygenase-1 and cyclooxygenase-2, as well as terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay, immunohistochemistry for heat-shock protein 70, B-cell lymphoma-2 and Bax were conducted. Results indicated that rats pretreated with pogostone showed remarkable protection from the gastric mucosa damage compared to vehicle-treated rats based on the ulcer index and inhibition percentage. Histologically, oral administration of pogostone resulted in observable improvement of gastric injury, characterized by reduction of necrotic lesion, flattening of gastric mucosa and alleviation of submucosal edema with hemorrhage. Pogostone pretreatment significantly raised the depressed activities of superoxide dismutase, glutathione and catalase, while reduced the elevated malonaldehyde level compared with indomethacin-induced group. Pogostone-pretreated group induced a significant increase in gastric mucosal prostaglandin E2 level and obvious up-regulation of protein levels and mRNA expressions of cyclooxygenase-1 and cyclooxygenase-2. Furthermore, antiapoptotic effect of pogostone was verified by terminal deoxynucleotidyltransferase-mediated dUTP

  6. Two Distinct Pathways Support Gene Correction by Single-Stranded Donors at DNA Nicks

    Directory of Open Access Journals (Sweden)

    Luther Davis

    2016-11-01

    Full Text Available Nicks are the most common form of DNA damage. The mechanisms of their repair are fundamental to genomic stability and of practical importance for genome engineering. We define two pathways that support homology-directed repair by single-stranded DNA donors. One depends upon annealing-driven strand synthesis and acts at both nicks and double-strand breaks. The other depends upon annealing-driven heteroduplex correction and acts at nicks. Homology-directed repair via these pathways, as well as mutagenic end joining, are inhibited by RAD51 at nicks but largely independent of RAD51 at double-strand breaks. Guidelines for coordinated design of targets and donors for gene correction emerge from definition of these pathways. This analysis further suggests that naturally occurring nicks may have significant recombinogenic and mutagenic potential that is normally inhibited by RAD51 loading onto DNA, thereby identifying a function for RAD51 in maintenance of genomic stability.

  7. To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.

    Directory of Open Access Journals (Sweden)

    Samantha S Katz

    Full Text Available Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB, or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293 cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.

  8. Siim Nestor soovitab : Jus Fine ja DJ Hype. Mutant ja Nick Luscombe. Kuurorti kogumik / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2005-01-01

    Inglise drum'n'bass-staar DJ Hype 17. märtsil klubis Privé üritusel "Jus' Fine". Inglise diskor Nick Luscombe üritusel "Mutant" 18. märtsil klubis Privé. Plaadifirma Kuurortrecords esitleb 18. märtsil oma uut kogumikplaati Pirita jõe suudmes asuval laeval Monica

  9. Nicking enzyme-assisted biosensor for Salmonella enteritidis detection based on fluorescence resonance energy transfer.

    Science.gov (United States)

    Song, Yang; Li, Wenkai; Duan, Yingfen; Li, Zhongjie; Deng, Le

    2014-05-15

    Salmonella enteritidis (S. enteritidis) outbreaks continue to occur, and have increased public awareness of this pathogen. Nicking endonuclease Nb.BbvC I is widely used for the detection of biomolecules and displays activity for specific double-stranded DNA (dsDNA). In this study, we developed a biosensor to detect S. enteritidis based on fluorescence resonance energy transfer (FRET) using nicking enzyme and carbon nanoparticles (CNPs). Because of the quenching effect of black hole quencher 1 (BHQ 1), the CNPs do not fluoresce in the reaction system. When the target bacteria are added, the nicking enzyme recognizes and cleaves the dsDNA fabricated by the interaction between probe and target. As a result, the CNPs dissociate from BHQ 1 and emit strong fluorescence. Using the nicking enzyme, the fluorescence signals of the biosensor are greatly amplified. The biosensor exhibited a linear relationship with the concentration of S. enteritidis ranging from 10(2) to 3 × 10(3)CFU/mL in water and from 1.5 × 10(2) to 3 × 10(3)CFU/mL in milk. The present results indicate that our FRET-based detection system can be widely employed for the effective detection of pathogens.

  10. Siim Nestor soovitab : Jus Fine ja DJ Hype. Mutant ja Nick Luscombe. Kuurorti kogumik / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2005-01-01

    Inglise drum'n'bass-staar DJ Hype 17. märtsil klubis Privé üritusel "Jus' Fine". Inglise diskor Nick Luscombe üritusel "Mutant" 18. märtsil klubis Privé. Plaadifirma Kuurortrecords esitleb 18. märtsil oma uut kogumikplaati Pirita jõe suudmes asuval laeval Monica

  11. The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism.

    Science.gov (United States)

    Gaillard, Pierre-Henri L; Noguchi, Eishi; Shanahan, Paul; Russell, Paul

    2003-09-01

    Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.

  12. Perspectives on the ‘lens of risk’ interview series: interview with Nick Pidgeon

    NARCIS (Netherlands)

    Heyman, B.; Brown, P.

    2012-01-01

    This article is the first in a series which will appear in 2012 in the special issue series Health Care Through the `Lens of Risk'. It provides a quasi-verbatim transcript of an interview with Nick Pidgeon, one of the main contributors to the social science component of The Royal Society Risk Report

  13. Desvenlafaxine reduces apoptosis in amygdala after myocardial infarction.

    Science.gov (United States)

    Malick, Mandy; Gilbert, Kim; Barry, Mathieu; Godbout, Roger; Rousseau, Guy

    2014-10-01

    This study was designed to determine if desvenlafaxine (DV), a serotonin-norepinephrine reuptake inhibitor, can attenuate apoptosis observed in the limbic system after myocardial infarction (MI). MI was induced in rats by occlusion of the left descending artery for 40 min followed by reperfusion. Another group of sham (control) rats was similarly manipulated, but without occlusion. Half of the full cohort received DV (3 mg/kg/day intraperitoneal), starting 5 min after the onset of reperfusion; the other half received the vehicle (0.5 ml of 0.9% saline). Rats were sacrificed after 3 days for biochemical analyses and MI size measurements. Infarct size was significantly smaller in DV- compared to vehicle-treated rats. At 3 days post-MI, caspase-3 and -8 activities and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive cells were decreased in the amygdala of DV-treated rats compared to MI-vehicle controls. No difference was observed between the sham groups. The data indicates that DV given immediately after an acute MI event can reduce MI size and apoptosis in amygdala when measured three days post-MI. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Nick Hornby’s A Long Way Down (2005 in the Transition from Book to Movie

    Directory of Open Access Journals (Sweden)

    Ömercan Tüm

    2015-12-01

    Full Text Available Nick Hornby’s A Long Way Down (2005 in the Transition from Book to movie Abstract Challenging and profound, Nick Hornby’s novel A Long Way Down (2005 is the story of four people failing to commit suicide. The protagonists are caught in an intricate web of relationships, disappointments and missed chances on their one-way journey to understand that “The cure for unhappiness is happiness” (Elizabeth McCracken. This paper aims at demonstrating that 2014 movie version directed by Pascal Chaumeil fails to capture the essence of the book and resorts to a number of radical changes which are only supposed to attract a larger audience, but do not necessarily send the same message as the novel.

  15. Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI.

    Directory of Open Access Journals (Sweden)

    Benjamin P Kleinstiver

    Full Text Available Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

  16. Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis

    Science.gov (United States)

    Khan, Sharik R.; Kuzminov, Andrei

    2013-01-01

    Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percent of chromosomal DNA entering the gel. The degree of separation in PFG depends upon the size of DNA, as well as various conditions of electrophoresis, such as electric field strength (FS), time of electrophoresis, switch time and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that sub-chromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single strand interruptions results in artefactual decrease in molecular weight of linear DNA making accurate determination of the number of double strand breaks difficult. While breakage of nicked sub-chromosomal fragments is FS-independent, some high molecular weight sub-chromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. PMID:23770235

  17. Trapping and breaking of in vivo nicked DNA during pulsed field gel electrophoresis.

    Science.gov (United States)

    Khan, Sharik R; Kuzminov, Andrei

    2013-12-15

    Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, time of electrophoresis, switch time, and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. "If I Were Nick": Men's Responses to an Interactive Video Drama Series to Support Smoking Cessation.

    Science.gov (United States)

    Bottorff, Joan L; Sarbit, Gayl; Oliffe, John L; Kelly, Mary T; Lohan, Maria; Stolp, Sean; Sharp, Paul

    2015-08-10

    Men continue to smoke in greater numbers than women; however, few interventions have been developed and tested to support men's cessation. Men tend to rely on quitting strategies associated with stereotypical manliness, such as willpower, stoicism, and independence, but they may lack the self-efficacy skills required to sustain a quit. In this paper, we describe the development of and reception to an interactive video drama (IVD) series, composed of 7 brief scenarios, to support and strengthen men's smoking cessation efforts. The value of IVD in health promotion is predicated on the evidence that viewers engage with the material when they are presented characters with whom they can personally identify. The video dramatizes the challenges unfolding in the life of the main character, Nick, on the first day of his quit and models the skills necessary to embark upon a sustainable quit. The objective was to describe men's responses to the If I were Nick IVD series as part of a study of QuitNow Men, an innovative smoking cessation website designed for men. Specific objectives were to explore the resonance of the main character of the IVD series with end-users and explore men's perceptions of the effectiveness of the IVD series for supporting their quit self-management. Seven brief IVD scenarios were developed, filmed with a professional actor, and uploaded to a new online smoking cessation website, QuitNow Men. A sample of 117 men who smoked were recruited into the study and provided baseline data prior to access to the QuitNow Men website for a 6-month period. During this time, 47 men chose to view the IVDs. Their responses to questions about the IVDs were collected in online surveys at 3-month and 6-month time points and analyzed using descriptive statistics. The majority of participants indicated they related to the main character, Nick. Participants who "strongly agreed" they could relate to Nick perceived significantly higher levels of support from the IVDs than the

  19. Real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons.

    Science.gov (United States)

    Xu, Wentao; Wang, Chenguang; Zhu, Pengyu; Guo, Tianxiao; Xu, Yuancong; Huang, Kunlun; Luo, Yunbo

    2016-04-21

    Techniques of isothermal amplification have recently made great strides, and have generated significant interest in the field of point-of-care detection. Nicking endonuclease-mediated isothermal amplification (NEMA) is an example of simple isothermal technology. In this paper, a real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons (SMB-NEMA) of improved specificity and sensitivity is described. First, we optimized the prohibition of de novo synthesis by choosing Nt·BstNBI endonuclease. Second, the whole genome was successfully amplified with Nt·BstNBI (6 U), betaine (1 M) and trehalose (60 mM) for the first time. Third, we achieved 10 pg sensitivity for the first time after adding a small molecular beacon that spontaneously undergoes a conformational change when hybridizing to target, and the practical test validated the assay's application. The small molecular beacon has a similar melting temperature to the reaction temperature, but is approximately 10 bp shorter than the length of a traditional molecular beacon. A new threshold regulation was also established for isothermal conditions. Finally, we established a thermodynamic model for designing small molecular beacons. This multistate model is more correct than the traditional algorithm. This theoretical and practical basis will help us to monitor SMB-NEMA in a quantitative way. In summary, our SMB-NEMA method allows the simple, specific and sensitive assessment of isothermal DNA quantification.

  20. Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling.

    Science.gov (United States)

    Quarrie, L H; Addey, C V; Wilde, C J

    1995-09-01

    Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.

  1. From High Fidelity (1995 to Funny Girl (2014 or What Makes Nick Hornby’s Novels so Popular

    Directory of Open Access Journals (Sweden)

    Cristina Chifane

    2015-12-01

    Full Text Available From High Fidelity (1995 to Funny Girl (2014 or What Makes Nick Hornby’s Novels so Popular Abstract  The common and perhaps the most fascinating characteristic of all Nick Hornby’s novels is that they tackle contemporary problems of ordinary people. As a consequence, the readers will plunge into a world of failed relationships; fear of commitment; depression; lack of emotional stability; teenage anger and imbalance; frustration and obsession; invented maladies, sons or parties; disappointment and self-pity as well as useless single or group therapies. In spite of their dark problematic, Hornby’s novels have enjoyed popularity and continue to exert the sort of fascination that only brilliant literary pieces may produce. From such a point of view, this paper will try to solve the puzzle called Nick Hornby constructing and deconstructing the elements that make up his unique writing style.

  2. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    Science.gov (United States)

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.

  3. Synapsis alters RAG-mediated nicking at Tcrb recombination signal sequences: implications for the “beyond 12/23” rule.

    Science.gov (United States)

    Banerjee, Joydeep K; Schatz, David G

    2014-07-01

    At the Tcrb locus, Vβ-to-Jβ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the “beyond 12/23” rule (B12/23 rule). Previous work showed that Vβ recombination signal sequences (RSSs) do not recombine with Jβ RSSs because Jβ RSSs are crippled for either nicking or synapsis. This result raised the following question: how can crippled Jβ RSSs recombine with Dβ RSSs? We report here that the nicking of some Jβ RSSs can be substantially stimulated by synapsis with a 3′Dβ1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of a partner. Slow-nicking RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial RAG binding can occur on RSSs with either 12- or 23-bp spacers (12- or 23-RSSs, respectively).

  4. Termination of packaging of the bacteriophage lambda chromosome: cosQ is required for nicking the bottom strand of cosN.

    Science.gov (United States)

    Cue, D; Feiss, M

    1998-07-03

    Termination of packaging of the lambda chromosome involves completion of translocation of the DNA into the head shell, and conversion of the translocation complex into a cleavage complex. The cleavage reaction introduces staggered nicks into the downstream cosN to generate the right cohesive end of the chromosome. cosQ, a site adjacent to cosN, was found to be required for nicking the bottom strand of cosN; bottom strand nicking was also sequence-specific for bps at the nick site. Nicking of the top strand of cosN (cosNL) was stimulated by cosQ, but fidelity and efficiency of cosNL nicking were largely dictated by other cos subsites (i.e. cosB and I2). Aberrant top-strand cleavage within cosQ was observed in the absence of I2, and nicking at a site 8 nt 5' to the normal cosNL nick site occurred in the absence of cosB. The presence of cosQ was found to be insufficient to arrest DNA translocation in vivo, indicating that cosQ, per se, is not a packaging stop signal. A model is presented in which the role of cosQ is to depolarize the asymmetric arrangement of terminase protomers in the translocation complex so that protomers are configured to match the 2-fold rotational symmetry of cosN.

  5. W. Horsley Gantt, Nick, and the Pavlovian Science at Phipps Clinic.

    Science.gov (United States)

    Ruiz, Gabriel; Sánchez, Natividad

    2016-10-24

    William Horsley Gantt is well known as one to the principal proponents of Pavlovian methodology in the U.S. After a long stay at Ivan Petrovich Pavlov's laboratory in Leningrad from 1925 to 1929, Gantt was invited by Adolf Meyer to join the Henry Phipps Psychiatric Clinic, where he founded and directed the Pavlovian Laboratory from 1930 to 1964. Soon after his arrival at Phipps Clinic in 1931, Gantt began a Pavlovian research program that included the investigation of nervous disturbances in dogs and clinical researches with psychiatric patients. In these studies, Gantt combined a physiological method (the conditional reflexes approach), with a psychiatric problem (nervous disorders) in the context of Meyer's psychobiology that established the person or individual as unit of analysis. This fact, concentrating upon a single individual, made Gantt studies with dogs recognizable and interesting to physicians, psychologists, and psychiatrists who also worked on individuals. In this paper, we use archival materials -including correspondence, notebooks, and unpublished autobiographical material- to present a case study, that of William Horsley Gantt and his dog Nick. We will explore the reasons why Gantt' studies on nervous disturbances with this dog captured the interest of psychiatrists and clinical psychologists.

  6. The Demystification of Nick Zangwill’s “Myth of Religious Experience”

    Directory of Open Access Journals (Sweden)

    Fasko Manuel

    2017-01-01

    Full Text Available The debate about religious experiences has recently been shaped by the question of whether they exist or if they are a myth. One of the most compelling arguments for the non-existence of religious experience was put forward by Nick Zangwill. In his “The myth of religious experience” (2004 he argued that God can be perceived neither by our ordinary five senses nor by some special sixth sense. While I agree with Zangwill that God cannot be perceived with our ordinary five senses (or a sixth religious sense, I do not think his argument shows that religious experience - based on Zangwill’s own understanding of the term - is a myth. In this paper, I offer in two steps a philosophical defence - in the analytical tradition - of the possible existence of religious experience as perceptual experiences. In the first step, I adumbrate Zangwill’s argument for the myth of religious experience, which fails because it ultimately begs the question - as I argue in the second step, by presenting a Berkelean answer to Zangwill’s challenge.

  7. Optimization of a DNA Nicking Assay to Evaluate Oenocarpus bataua and Camellia sinensis Antioxidant Capacity

    Directory of Open Access Journals (Sweden)

    Louis-Jérôme Leba

    2014-10-01

    Full Text Available This study was aimed at assessing the DNA damage protective activity of different types of extracts (aqueous, methanolic and acetonic using an in vitro DNA nicking assay. Several parameters were optimized using the pUC18 plasmid, especially FeSO4, EDTA, solvent concentrations and incubation time. Special attention has been paid to removing the protective and damaging effect of the solvent and FeSO4 respectively, as well as to identifying the relevant positive and negative controls. For each solvent, the optimal conditions were determined: (i for aqueous extracts, 0.33 mM of FeSO4 and 0.62 mM of EDTA were incubated for 20 min at 37 °C; (ii for acetone extracts, 1.16% solvent were incubated for 15 min at 37 °C with 1.3 mM of FeSO4 and 2.5 mM of EDTA and (iii for methanol extracts, 0.16% solvent, were incubated for 1.5 h at 37 °C with 0.33 mM of FeSO4 and 0.62 mM of EDTA. Using the optimized conditions, the DNA damage protective activity of aqueous, methanolic and acetonic extracts of an Amazonian palm berry (Oenocarpus bataua and green tea (Camellia sinensis was assessed. Aqueous and acetonic Oenocarpus bataua extracts were protective against DNA damage, whereas aqueous, methanolic and acetonic extracts of Camellia sinensis extracts induced DNA damage.

  8. Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Villeponteau, B.; Martinson, H.G.

    1987-05-01

    The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA. This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

  9. The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction.

    Science.gov (United States)

    Cue, D; Feiss, M

    1993-12-05

    cosB is the binding site for terminase, the DNA packaging enzyme of ai-12581mbda, and cosN is the adjacent site at which terminase gm-07228es staggered nicks to generate mature lambda DNA molecules. There are three binding sites (R3, R2 and R1) within cosB for gpNu1, the small subunit of terminase. A particular transition mutation of R1, known to weaken binding of gpNu1 to R1, has been introduced into the other R sites, and in the present work the effects of R site mutations on nicking of cosN have been examined. Nicking experiments performed in the presence of ATP suggest that the most profound cosB mutation tested (the R3-R2-R1- mutation) would, at most, reduce cos nicking to congruent to 30% of the level observed for the wild-type substrate. In the presence of ATP, the R3-R2-R1- mutation had no significant effect on terminase nicking of the 1 strand and reduced r-strand nicking to 35% of the wild-type level. The other cosB mutations had no effect on the nicking of either DNA strand when nucleotide was added, but in the absence of ATP, most of the cos mutations resulted in some form of cosN nicking defect; the nicking defects, however, are milder than the in vivo packaging defects that result from the mutations. Quantitatively, only the effect of the R3-R2-R1- mutation on in vitro cosN nicking is reflective of the growth defect exhibited by a R3-R2-R1- phage but the nicking defect is only observed when ATP is omitted from the reaction. The proposal that the cosB mutations primarily affect DNA packaging rather than cosN nicking is discussed. All of the cosB mutations affect r-strand nicking to a greater extent than 1-strand nicking, implying that the interaction of terminase with the left half of cosN occurs via the direct recognition of cosNL by terminase. The level of DNA substrate required for half-maximal cos nicking is approximately equivalent for reactions performed in the presence or absence of ATP, indicating that ATP does not increase the affinity of

  10. Life or death by NFκB, Losartan promotes survival in dy2J/dy2J mouse of MDC1A

    Science.gov (United States)

    Elbaz, M; Yanay, N; Laban, S; Rabie, M; Mitrani-Rosenbaum, S; Nevo, Y

    2015-01-01

    Inflammation and fibrosis are well-defined mechanisms involved in the pathogenesis of the incurable Laminin α2-deficient congenital muscular dystrophy (MDC1A), while apoptosis mechanism is barely discussed. Our previous study showed treatment with Losartan, an angiotensin II type I receptor antagonist, improved muscle strength and reduced fibrosis through transforming growth factor beta (TGF-β) and mitogen-activated protein kinases (MAPK) signaling inhibition in the dy2J/dy2J mouse model of MDC1A. Here we show for the first time that Losartan treatment up-regulates and shifts the nuclear factor kappa B (NFκB) signaling pathway to favor survival versus apoptosis/damage in this animal model. Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-β protein level, indicating NFκB activation. Moreover, NFκB anti-apoptotic target genes TNF receptor-associated factor 1 (TRAF1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were increased following Losartan treatment. Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased. Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. Thus, along with TGF-β and MAPK signaling, NFκB serves as an important regulatory pathway which following Losartan treatment promotes survival in the dy2J/dy2J mouse model of MDC1A. PMID:25766329

  11. Garlic (Allium sativum Fresh Juice Induces Apoptosis in Human Oral Squamous Cell Carcinoma: The Involvement of Caspase-3, Bax and Bcl-2

    Directory of Open Access Journals (Sweden)

    Farrokh Farhadi

    2015-12-01

    Full Text Available Background and aims. There is no report on the apoptotic impact of Allium sativum L.(Garlic on the oral squamous cell carcinoma (KB; hence, this study was designed to survey the apoptotic effects of garlic fresh juice (GFJ on the KB cells. Materials and methods. MTTassay (MicrocultureTetrazolium Assay was carried out to evaluate the cytotoxicity of GFJ on KB cells. Furthermore, TUNEL(Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelingand DNA fragmentation tests were performed to determine if GFJ is able to induce apoptosis in KB cells. Also a standard kit was used to assess caspase-3 activity in KB cells. Also western blotting was employed to evaluate the effect of GFJ on Bax:Bcl-2 ratio. Results. Significant cytotoxic effects were observed for the minimum used concentration (1g/mL as calculated to be 77.97±2.3% for 24 h and 818±3.1% for 36h of incubation (P < 0.001. Furthermore, TUNEL and DNA fragmentation tests corroborated the apoptosis inducing activity of GFJ. Consistently, after treating KB cells with GFJ(1μg/mL, caspase-3 activity and Bax:Bcl-2 ratio were raised by 7.3±0.6 and (P <0.001 folds, respectively. Conclusion. The results of this study advanced that GFJ induces apoptosis in the KB cells through increasing caspase-3 activity and Bax:Bcl2 ratio which could be attributed to its organo-sulfurcomponents.

  12. A Preliminary Study: N-acetyl-L-cysteine Improves Semen Quality following Varicocelectomy

    Directory of Open Access Journals (Sweden)

    Foroogh Barekat

    2016-05-01

    Full Text Available Background: Surgery is considered the primary treatment for male infertility from clinical varicocele. One of the main events associated with varicocele is excessive production of reactive oxygen species (ROS. N-acetyl-L-cysteine (NAC, an antioxidant that scavenges free radicals, is considered a supplement to alleviate glutathione (GSH depletion during oxidative stress. Despite beneficial effects of NAC in other pathological events, there is no report on the effect of NAC in individuals with varicocele. Therefore, the aim of this study is to evaluate the outcome of NAC on semen quality, protamine content, DNA damage, oxidative stress and fertility following varicocelectomy. Materials and Methods: This prospective clinical trial included 35 infertile men with varicocele randomly divided into control (n=20 and NAC (n=15 groups. We assessed semen parameters, protamine content [chromomycin A3 (CMA3], DNA integrity [terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL] and oxidative stress [2', 7'-dichlorodihydrofluorescein-diacetate (DCFH-DA] before and three months after varicocelectomy. Results: Percentage of abnormal semen parameters, protamine deficiency, DNA fragmentation and oxidative stress were significantly decreased in both groups compared to before surgery. We calculated the percentage of improvement in these parameters compared to before surgery for each group, then compared the results between the groups. Only percentage of protamine deficiency and DNA fragmentation significantly differed between the NAC and control groups. Conclusion: The results of this study, for the first time, revealed that NAC improved chromatin integrity and pregnancy rate when administered as adjunct therapy post-varicocelectomy (Registeration Number: IRCT201508177223N5.

  13. Influence of heat stress to matrix on bone formation.

    Science.gov (United States)

    Yoshida, Keiko; Uoshima, Katsumi; Oda, Kimimitsu; Maeda, Takeyasu

    2009-08-01

    It is important to know the etiology of implant failure. It has been reported that heat stress during drilling was one of the causes for failure and the threshold was 47 degrees C. However, clinically, we encounter cases in which overheating does not seem to affect osseointegration eventually. The purpose of this study was to assess histologically the spatio-temporal effect of heat stress on bone formation after overheating the bone matrix. Rat calvarial bone was heated to 37 degrees C, 43 degrees C, 45 degrees C and 48 degrees C for 15 min by a temperature stimulator. Paraffin sections were prepared 1, 3 and 5 weeks after heating and investigated histologically under light microscopy. Hematoxylin and eosin staining, alkaline phosphatase (ALP), osteopontin (OPN), heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were carried out. The area of dead osteocytes was calculated and statistically analyzed. Apoptotic osteocytes were detected by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method. Along with the temperature increase, the area of dead osteocytes increased and regeneration of the periosteal membrane was delayed. Hsps- and TUNEL-positive cells were only seen in the 48 degrees C group. Spatio-temporal changes of TRAP- and ALP-positive cell numbers were observed, while OPN expression was mostly absent. Even after 48 degrees C stimulation, bone formation on the calvarial surface was observed after 5 weeks. Although there was a temperature-dependent delay in bone formation after heat stress, the 48 degrees C heat stress did not obstruct bone formation eventually. This delay was probably caused by slow periosteal membrane regeneration.

  14. Trichomonas vaginalis induces cytopathic effect on human lung alveolar basal carcinoma epithelial cell line A549.

    Science.gov (United States)

    Salvador-Membreve, Daile Meek C; Jacinto, Sonia D; Rivera, Windell L

    2014-12-01

    Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association.

  15. Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Ricardo Chaves Vilela

    2012-09-01

    Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such

  16. Inhibition of signal transducer and activator of transcription 3 expression by RNA interference suppresses invasion through inducing anoikis in human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yu Fan; You-Li Zhang; Ying Wu; Wei Zhang; Yin-Huan Wang; Zhao-Ming Cheng; Hua Li

    2008-01-01

    AIM: To investigate the roles and mechanism of signal transducer and activator of transcription 3 (STAT3) in invasion of human colon cancer cells by RNA interference. METHODS: Small interfering RNA (siRNA) targeting Signal transducer and activator of transcription 3 (STAT3) was transfected into HT29 colon cancer cells. STAT3 protein level and DNA-binding activity of STAT3 was evaluated by western blotting and electrophoretic mobility shift assay (EMSA), respectively. We studied the anchorage-independent growth using colony formation in soft agar, and invasion using the boyden chamber model, anoikis using DNA fragmentation assay and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL), respectively. Western blot assay was used to observe the protein expression of Bcl-xL and survivin in colon cancer HT29 cells. RESULTS: RNA interference (RNAi) mediated by siRNA leads to suppression of STAT3 expression in colon cancer cell lines. Suppression of STAT3 expression by siRNA could inhibit anchorage-independent growth, and invasion ability, and induces anoikis in the colon cancer cell line HT29. It has been shown that knockdown of STAT3 expression by siRNA results in a reduction in expression of Bcl-xL and survivin in HT29 cells. CONCLUSION: These results suggest that STAT3 siRNA can inhibit the invasion ability of colon cancer cells through inducing anoikis, which antiapoptotic genes survivin and Bcl-xL contribute to regulation of anoikis. These studies indicate STAT3 siRNA could be a useful therapeutic tool for the treatment of colon cancer.

  17. Sulindac induces apoptosis and protects against colon carcinoma in mice

    Institute of Scientific and Technical Information of China (English)

    Bao-Cun Sun; Xiu-Lan Zhao; Shi-Wu Zhang; Yi-Xin Liu; Lan Wang; Xin Wang

    2005-01-01

    AIM: To study the effect of sulindac on colon cancer induction in mice.METHODS: The chemo-preventive action of 80 ppm sulindac fed during initiation and post-initiation and 100 ppm sulindac fed during progressive stages of induction of colon carcinogenesis in mice was investigated using 1,2-dimethylhydrazine (DMH). Using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)technique and PCNA immunohistochemical staining, we observed the apoptotic and proliferative cell density changes at different carcinogenic stages and the effect of sulindac on these two phenomena.RESULTS: Dietary sulindac significantly inhibited the incidence of colonic neoplasmas in mice. Compared with the control group, feeding sulindac during initiation and post-initiation stages inhibited the incidence by 46.7-50.4%,and feeding sulindac during progressive stages inhibited the incidence by 41.1%. Animals that were fed sulindac showed less serious pathological changes than those that were fed the control diet (P<0.01, H= 33.35). There was no difference in the density of proliferating cells among those groups which were or were not fed sulindac. In the same period, feeding sulindac resulted in a higher density of apoptotic cells than feeding control diet. CONCLUSION: Sulindac has an anti-carcinogenic function in mice. Its effect on preventing colon carcinogenesis is better than its effect on treating established tumors. By inducing apoptosis, sulindac inhibited the development of colon cancer and delayed canceration. Sulindac has no effect on proliferation. The anti-carcinogenic properties of sulindac are most effective in the moderate and severe stages of dysplasia and canceration.

  18. Sensitive and specific colorimetric DNA detection by invasive reaction coupled with nicking endonuclease-assisted nanoparticles amplification.

    Science.gov (United States)

    Zou, Bingjie; Cao, Xiaomei; Wu, Haiping; Song, Qinxin; Wang, Jianping; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

    2015-04-15

    Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.

  19. Human MUS81-EME2 can cleave a variety of DNA structures including intact Holliday junction and nicked duplex.

    Science.gov (United States)

    Amangyeld, Tamir; Shin, Yong-Keol; Lee, Miju; Kwon, Buki; Seo, Yeon-Soo

    2014-05-01

    MUS81 shares a high-degree homology with the catalytic XPF subunit of the XPF-ERCC1 endonuclease complex. It is catalytically active only when complexed with the regulatory subunits Mms4 or Eme1 in budding and fission yeasts, respectively, and EME1 or EME2 in humans. Although Mus81 complexes are implicated in the resolution of recombination intermediates in vivo, recombinant yeast Mus81-Mms4 and human MUS81-EME1 isolated from Escherichia coli fail to cleave intact Holliday junctions (HJs) in vitro. In this study, we show that human recombinant MUS81-EME2 isolated from E. coli cleaves HJs relatively efficiently, compared to MUS81-EME1. Furthermore, MUS81-EME2 catalyzed cleavage of nicked and gapped duplex deoxyribonucleic acids (DNAs), generating double-strand breaks. The presence of a 5' phosphate terminus at nicks and gaps rendered DNA significantly less susceptible to the cleavage by MUS81-EME2 than its absence, raising the possibility that this activity could play a role in channeling damaged DNA duplexes that are not readily repaired into the recombinational repair pathways. Significant differences in substrate specificity observed with unmodified forms of MUS81-EME1 and MUS81-EME2 suggest that they play related but non-overlapping roles in DNA transactions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Enhanced nicking activity of Rep in presence of pre-coat protein of Mungbean yellow mosaic India virus.

    Science.gov (United States)

    Rouhibakhsh, A; Choudhury, N R; Mukherjee, S K; Malathi, V G

    2012-04-01

    Yellow mosaic disease causes severe yield loss in grain legumes in Indian subcontinent and south east Asia. The disease is caused by two virus species, Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV). They have genome organization typical of Old World begomoviruses, the unique feature being the presence of an open reading frame (ORF) AV2 upstream of coat protein gene. In order to elucidate its function, ORF AV2 of blackgram isolate, Mungbean yellow mosaic India virus-[India:New Delhi:Blackgram 3:1991] MYMIV-[IN:ND:Bg3:91] and cowpea isolate, Mungbean yellow mosaic India virus-[India:New Delhi:Cowpea7:1998] MYMIV-[IN:ND:Cp7:98], respectively, were over expressed in Escherichia coli in fusion with maltose binding protein (MBP). The recombinant protein did not show efficient binding to DNA. However, both MBP-BgAV2 and MBP-CpAV2 proteins modulated nicking and ATPase activity of replication initiation protein (Rep). Even low concentration, 20 ng of MBP-BgAV2 and MBP-CpAV2 could bring 20 folds increase in nicking activity of Rep. Similarly in the presence of AV2 protein, two to three fold increase in ATPase activity was observed. It is hypothesized that AV2 protein may play a role of accessory protein modulating Rep activities.

  1. Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Takuya Sato

    2015-07-01

    Full Text Available Mouse spermatogonial stem cells (SSCs can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure.

  2. Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.

    Science.gov (United States)

    Renouf, Benjamin; Piganeau, Marion; Ghezraoui, Hind; Jasin, Maria; Brunet, Erika

    2014-01-01

    Recurrent chromosomal translocations are found in numerous tumor types, often leading to the formation and expression of fusion genes with oncogenic potential. Creating chromosomal translocations at the relevant endogenous loci, rather than ectopically expressing the fusion genes, opens new possibilities for better characterizing molecular mechanisms driving tumor formation. In this chapter, we describe methods to create cancer translocations in human cells. DSBs or paired nicks generated by either wild-type Cas9 or the Cas9 nickase, respectively, are used to induce translocations at the relevant loci. Using different PCR-based methods, we also explain how to quantify translocation frequency and to analyze breakpoint junctions in the cells of interest. In addition, PCR detection of translocations is used as a very sensitive method to detect off-target effects, which has general utility.

  3. Synapsis Alters RAG-Mediated Nicking at Tcrb Recombination Signal Sequences: Implications for the “Beyond 12/23” Rule

    OpenAIRE

    Banerjee, Joydeep K.; Schatz, David G.

    2014-01-01

    At the Tcrb locus, Vβ-to-Jβ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the “beyond 12/23” rule (B12/23 rule). Previous work showed that Vβ recombination signal sequences (RSSs) do not recombine with Jβ RSSs because Jβ RSSs are crippled for either nicking or synapsis. This result raised the following question: how can crippled Jβ RSSs recombine with Dβ RSSs? We report here that the nicking of some Jβ RSSs can be substantially stimulated by sy...

  4. John Steinbeck: "The Pearl," Adapted by Warren Frost and Dramatized for the Kennedy Center by Nick Olcott. Cue Sheet for Students.

    Science.gov (United States)

    Carr, John C.

    This performance guide is designed for teachers to use with students before and after a performance of "The Pearl" by John Steinbeck, adapted by Warren Frost and dramatized for the Kennedy Center by Nick Olcott. It is in the form of a Director's Notebook--a scrapbook/journal of clippings, memos, lists, illustrations, notes, and other…

  5. DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism.

    Science.gov (United States)

    Gubaev, Airat; Weidlich, Daniela; Klostermeier, Dagmar

    2016-12-01

    The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. We propose an alternative mechanism for DNA supercoiling via nicking and closing of DNA that involves trapping, segregation and relaxation of two positive supercoils. In contrast to DNA supercoiling, ATP-dependent relaxation and decatenation of DNA by gyrase lacking the C-terminal domains require both tyrosines and strand passage. Our results point towards mechanistic plasticity of gyrase and might pave the way for finding novel and specific mechanism-based gyrase inhibitors. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Enhancement of RecA-mediated self-assembly in DNA nanostructures through basepair mismatches and single-strand nicks

    Science.gov (United States)

    Corbett, Sybilla Louise; Sharma, Rajan; Davies, Alexander Giles; Wälti, Christoph

    2017-01-01

    The use of DNA as a structural material for nanometre-scale construction has grown extensively over the last decades. The development of more advanced DNA-based materials would benefit from a modular approach enabling the direct assembly of additional elements onto nanostructures after fabrication. RecA-based nucleoprotein filaments encapsulating short ssDNA have been demonstrated as a tool for highly efficient and fully programmable post-hoc patterning of duplex DNA scaffold. However, the underlying assembly process is not fully understood, in particular when patterning complex DNA topologies. Here, we report the effect of basepair-mismatched regions and single-strand nicks in the double-stranded DNA scaffold on the yield of RecA-based assembly. Significant increases in assembly yield are observed upon the introduction of unpaired basepairs directly adjacent to the assembly region. However, when the unpaired regions were introduced further from the assembly site the assembly yield initially decreased as the length of the unpaired region was increased. These results suggest that an unpaired region acts as a kinetic trap for RecA-based nucleoprotein filaments, impeding the assembly mechanism. Conversely, when the unpaired region is located directly adjacent to the assembly site, it leads to an increase in efficiency of RecA patterning owing to increased breathing of the assembly site.

  7. Book Reviews / Nick Ellison

    Index Scriptorium Estoniae

    Ellison, Nick

    2008-01-01

    J. Aidukaite doktoridissertatsiooni, The Emergence of the Post-Socialist Welfare State - The Case of the Baltic States: Estonia, Latvia and Lithuania, milles võrreldakse nende riikide sotsiaalpoliitikat ja sotsiaalabi süsteeme, refereering

  8. Book Reviews / Nick Ellison

    Index Scriptorium Estoniae

    Ellison, Nick

    2008-01-01

    J. Aidukaite doktoridissertatsiooni, The Emergence of the Post-Socialist Welfare State - The Case of the Baltic States: Estonia, Latvia and Lithuania, milles võrreldakse nende riikide sotsiaalpoliitikat ja sotsiaalabi süsteeme, refereering

  9. The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination.

    Science.gov (United States)

    Drejer-Teel, Anna H; Fugmann, Sebastian D; Schatz, David G

    2007-09-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dbeta gene segment in the assembled T-cell receptor (TCR) beta variable region exon and is manifest by a failure of direct Vbeta-to-Jbeta gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRbeta gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRbeta locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRbeta locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jbeta substrates and to some extent through poor synapsis of Vbeta and Jbeta substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jbeta substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo.

  10. The Beyond 12/23 Restriction Is Imposed at the Nicking and Pairing Steps of DNA Cleavage during V(D)J Recombination▿

    Science.gov (United States)

    Drejer-Teel, Anna H.; Fugmann, Sebastian D.; Schatz, David G.

    2007-01-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dβ gene segment in the assembled T-cell receptor (TCR) β variable region exon and is manifest by a failure of direct Vβ-to-Jβ gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRβ gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRβ locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRβ locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jβ substrates and to some extent through poor synapsis of Vβ and Jβ substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jβ substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo. PMID:17636023

  11. The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB.

    Science.gov (United States)

    Hang, J Q; Catalano, C E; Feiss, M

    2001-11-06

    cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB.

  12. Fluorescence biosensing strategy based on mercury ion-mediated DNA conformational switch and nicking enzyme-assisted cycling amplification for highly sensitive detection of carbamate pesticide.

    Science.gov (United States)

    Wang, Xiuzhong; Hou, Ting; Dong, Shanshan; Liu, Xiaojuan; Li, Feng

    2016-03-15

    Pesticides are of great importance in agricultural and biological fields, but pesticide residues may harm the environment and human health. A highly sensitive fluorescent biosensor for the detection of carbamate pesticide has been developed based on acetylcholinesterase (AChE)-catalyzed hydrolysis product triggered Hg(2+) release coupled with subsequent nicking enzyme-induced cleavage of a duplex DNA for cycling amplification. In this protocol, two DNA probes, an unmodified single-stranded helper DNA probe 1 (HP1) and a quencher-fluorophore probe (QFP) are ingeniously designed. HP1 can be folded into hairpin configuration through T-Hg(2+)-T base pair formation. QFP, labeled with FAM and BHQ1 at its two terminals, contains the recognition sequence and the cleavage site of the nicking enzyme. In the presence of carbamate pesticide, the activity of AChE is inhibited, and the amount of the product containing the thiol group generated by the hydrolysis reaction of acetylthiocholine chloride (ACh) decreases, resulting in the release of a low concentration of Hg(2+). The number of HP1 that can be selectively unfolded would be reduced and the subsequent nicking enzyme-assisted cleavage processes would be affected, resulting in decreased fluorescence signals. The fluorescence intensity further decreases with the increase of the pesticide concentration. Therefore, the pesticide content can be easily obtained by monitoring the fluorescence signal change, which is inversely proportional to the logarithm of the pesticide concentration. The detection limit of aldicarb, the model analyte, is 3.3 μgL(-1), which is much lower than the Chinese National Standards or those previously reported. The as-proposed method has also been applied to detect carbamate pesticide residues in fresh ginger and artificial lake water samples with satisfactory results, which demonstrates that the method has great potential for practical application in biological or food safety field.

  13. Avatars de Napalm Girl, June 8, 1972 (Nick Ut) : variations autour d’une icône de la Guerre du Vietnam

    OpenAIRE

    LESME, Anne

    2015-01-01

    International audience; This essay examines and analyses the iconic status of Nick Ut’s Vietnam photograph Napalm Girl (“Vietnam Napalm”, 1972) and its recycling in postmodern art and various media. Since it was published, this image has stood for the trauma of the Vietnam War and its lasting effects on the American collective memory. The symbolic crucifixion of the naked child is all the more powerful as the spectator, in this decisive moment, is facing horror and is invited to act. From the...

  14. Label-free and ratiometric detection of nuclei acids based on graphene quantum dots utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme.

    Science.gov (United States)

    Wang, Guang-Li; Fang, Xin; Wu, Xiu-Ming; Hu, Xue-Lian; Li, Zai-Jun

    2016-07-15

    Herein, we report a ratiometric fluorescence assay based on graphene quantum dots (GQDs) for the ultrasensitive DNA detection by coupling the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for cascade signal amplifications. With o-phenylenediamine acted as the substrate of G-quadruplex/hemin DNAzyme, whose oxidization product (that is, 2,3-diaminophenazine, DAP) quenched the fluorescence intensity of GQDs (at 460nm) obviously, accompanied with the emergence of a new emission of DAP (at 564nm). The ratiometric signal variations at the emission wavelengths of 564 and 460nm (I564/I460) were utilized for label-free, sensitive, and selective detection of target DNA. Utilizing the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for amplified cascade generation of DAP, the proposed bioassay exhibited high sensitivity toward target DNA with a detection limit of 30fM. The method also had additional advantages such as facile preparation and easy operation.

  15. Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

    Science.gov (United States)

    Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

    2013-04-01

    Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

  16. Synergistic requirement of orphan nonamer-like elements and DNA bending enhanced by HMGB1 for RAG-mediated nicking at cryptic 12-RSS but not authentic 12-RSS.

    Science.gov (United States)

    Numata, Masashi; Nagata, Kyosuke

    2011-08-01

    V(D)J recombination is initiated by the specific binding of the recombination activating gene (RAG) complex to the heptamer and nonamer elements within recombination signal sequence (RSS). The break points associated with some chromosomal translocations contain cryptic RSSs, and mistargeting of RAG proteins to these less conserved elements could contribute to an aberrant V(D)J recombination. Recently, we found RAG-dependent recombination in the hotspots of TEL-AML1 t(12;21)(p13;q22) chromosomal translocation by an extrachromosomal recombination assay. Here, we describe using in vitro cleavage assays that RAG proteins directly bind to and introduce nicks into TEL and AML1 translocation regions, which contain several heptamer-like sequences. The cryptic nicking site within the TEL fragment was cleaved by RAG proteins essentially depending on a 12-RSS framework, and the nicking activity was enhanced synergistically by both HMGB1 and orphan nonamer-like (NL) sequences, which do not possess counterpart heptamers. In addition, we found that DNA bending stimulated by HMGB1 is indispensable for the HMGB1- and orphan NL element-dependent enhancement of RAG-mediated nicking at the cryptic 12-RSS. Collectively, we would propose the mechanism of HMGB1-dependent enhancement of RAG-mediated nicking at a cryptic RSS through enhanced DNA bending. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  17. Avatars de Napalm Girl, June 8, 1972 (Nick Ut) : variations autour d’une icône de la Guerre du Vietnam

    OpenAIRE

    Lesme, Anne

    2015-01-01

    Cet article s’attache à analyser les remplois et détournements issus de la photographie emblématique Napalm Girl de Nick Ut pendant la Guerre du Vietnam, dans des productions artistiques et différents médias. Dès sa publication, cette image a représenté le traumatisme de la Guerre du Vietnam et ses effets durables dans la mémoire collective américaine. L’effet de la crucifixion symbolique de l’enfant nu est d’autant plus puissant que le spectateur, dans cet instant décisif, fait face à l’horr...

  18. On the Tragedy of Nick in Snowdrops-The Tension between the Sophis-ticated and the Naïve

    Institute of Scientific and Technical Information of China (English)

    任潇潇

    2013-01-01

    Snowdrops is a tale of an Englishman abroad. Nick is a thirty-something lawyer working in Moscow to broker huge deals between banks, oil companies and property developers. His project on a major loan to a subsidiary of an energy company is underway, and meanwhile, he comes across an enchanting girl, Masha and soon becomes involved with Masha ’s world, psycho-logically. This thesis tries to analyze the tension of the conflict between the corrupted native and the naïve foreigner, and the so-phisticated disposition of the narrator and the other innocent part. And thorough analyzing the tension reflected from other char-acters, the reason for Nick’s tragedy is revealed.

  19. Nicking endonuclease-assisted signal amplification of a split molecular aptamer beacon for biomolecule detection using graphene oxide as a sensing platform.

    Science.gov (United States)

    Li, Xiang; Ding, Xuelian; Fan, Jing

    2015-12-07

    Sensitive and selective detection of ultralow concentrations of specific biomolecules is important in early clinical diagnoses and biomedical applications. Many types of aptasensors have been developed for the detection of various biomolecules, but usually suffer from false positive signals and high background signals. In this work, we have developed an amplified fluorescence aptasensor platform for ultrasensitive biomolecule detection based on enzyme-assisted target-recycling signal amplification and graphene oxide. By using a split molecular aptamer beacon and a nicking enzyme, the typical problem of false positive signals can be effectively resolved. Only in the presence of a target biomolecule, the sensor system is able to generate a positive signal, which significantly improves the selectivity of the aptasensor. Moreover, using graphene oxide as a super-quencher can effectively reduce the high background signal of a sensing platform. We select vascular endothelial growth factor (VEGF) and adenosine triphosphate (ATP) as model analytes in the current proof-of-concept experiments. It is shown that under optimized conditions, our strategy exhibits high sensitivity and selectivity for the quantification of VEGF and ATP with a low detection limit (1 pM and 4 nM, respectively). In addition, this biosensor has been successfully utilized in the analysis of real biological samples.

  20. A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex

    Directory of Open Access Journals (Sweden)

    Fukang Luo

    2015-01-01

    Full Text Available In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease-assisted target recycling amplification (TRA, rolling circle amplification (RCA and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form “Y” junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O82− system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

  1. I-PfoP3I: a novel nicking HNH homing endonuclease encoded in the group I intron of the DNA polymerase gene in Phormidium foveolarum phage Pf-WMP3.

    Directory of Open Access Journals (Sweden)

    Shuanglei Kong

    Full Text Available Homing endonucleases encoded in a group I self-splicing intron in a protein-coding gene in cyanophage genomes have not been reported, apart from some free-standing homing edonucleases. In this study, a nicking DNA endonuclease, I-PfoP3I, encoded in a group IA2 intron in the DNA polymerase gene of a T7-like cyanophage Pf-WMP3, which infects the freshwater cyanobacterium Phormidium foveolarum is described. The Pf-WMP3 intron splices efficiently in vivo and self-splices in vitro simultaneously during transcription. I-PfoP3I belongs to the HNH family with an unconventional C-terminal HNH motif. I-PfoP3I nicks the intron-minus Pf-WMP3 DNA polymerase gene more efficiently than the Pf-WMP4 DNA polymerase gene that lacks any intervening sequence in vitro, indicating the variable capacity of I-PfoP3I. I-PfoP3I cleaves 4 nt upstream of the intron insertion site on the coding strand of EXON 1 on both intron-minus Pf-WMP3 and Pf-WMP4 DNA polymerase genes. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a 14 bp region downstream of the cut site. I-PfoP3I requires Mg(2+, Ca(2+ or Mn(2+ for nicking activity. Phylogenetic analysis suggests that the intron and homing endonuclease gene elements might be inserted in Pf-WMP3 genome individually after differentiation from Pf-WMP4. To our knowledge, this is the first report of the presence of a group I self-splicing intron encoding a functional homing endonuclease in a protein-coding gene in a cyanophage genome.

  2. Detection of apoptosis in human preimplantation embryos by TdT-mediated nick end labeling%人植入前胚胎细胞凋亡的细胞化学检测

    Institute of Scientific and Technical Information of China (English)

    陈雯; 王雁林; 胡娟; 朱桂金

    2006-01-01

    目的建立原位末端标记法(TdT-mediated nick end labeling,TUNEL)检测人植入前胚胎凋亡征象的方法.方法取试管婴儿助孕技术后异常受精的3原核胚胎,于2~10细胞期进行固定,采用TUNEL法检测3原核胚胎中的凋亡征象.结果在发育正常和发育停滞胚胎中,未检测到凋亡征象;在20%有碎片的胚胎中检测到TUNEL阳性信号.结论采用TUNEL法可以有效检测人植入前胚胎中的凋亡征象,细胞碎片的出现可能与凋亡有关.

  3. Nick Faldo的完美切击

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ 背景:这是Faldo第8次代表欧洲队出征个人对抗赛,星期日与他捉对厮杀的是宿敌Curtis Strange.在1988年美国公开赛上,Faldo在最后4洞吞了3个柏忌,并在加洞赛上不敌Strange,将冠军拱手相让.赛事逐渐白热化,很快大家都意识到,这局关乎本届莱德杯胜负的比洞赛将会非常艰苦.Strange在第11洞捉到一只小鸟,从而取得微弱的优势,及至第17洞,他仅以一洞的优势领先.

  4. Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis

    Science.gov (United States)

    de la Rosa, Enrique J.; Vega-Núñez, Elena; Morales, Aixa V.; Serna, José; Rubio, Eva; de Pablo, Flora

    1998-01-01

    Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis. PMID:9707581

  5. Apoptosis and autophagy induction in mammalian cells by small interfering RNA knockdown of mRNA capping enzymes.

    Science.gov (United States)

    Chu, Chun; Shatkin, Aaron J

    2008-10-01

    Addition of a 5' cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK(-/-) BAX(-/-) double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.

  6. Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms.

    Science.gov (United States)

    Jackson, Desmond N; Yang, Lin; Wu, ShiBiao; Kennelly, Edward J; Lipke, Peter N

    2015-10-01

    Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 μM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC50s) of 30 to 50 μM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC50 from >1,024 μg/ml to 13 μg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis.

  7. Enhancement of apoptosis by aspirin combined with sorafenib in a highly metastatic murine model of human hepatocellular carcinoma%阿司匹林抑制核因子-κB增强索拉非尼对肝癌的促凋亡作用

    Institute of Scientific and Technical Information of China (English)

    张伟; 孙惠川; 熊宇泉; 庄鹏远; 朱小东; 汤钊猷

    2011-01-01

    Objective To investigate the effect of combination of sorafenib and aspirin on tumor apoptosis,tumor growth,lung metastasis and survival of tumor-beating nude mice in a highly metastatic xenograft murine model of human hepatocellular carcinoma (HCC) and explore the mechanism.Methods The Xenograft of a highly metastatic human HCC tumor (LCI-D20) was used to evaluate survival,primary tumor growth and lung metastasis after treatment with aspirin alone or in combination with sorafenib.Apoptosis of tumor cells in vivo was determined by the terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling assay (TUNEL).Results Tumor volumes were (4.76 ±0.51 ),( 1.41 ±0.08),(0.66±0.12) and (4.58 ±0.47) cm3 in control,serafenib,serafenib plus aspirin,and aspirin groups,respectively.Lung metastases were 189 ±21,96 ± 15,30 ± 17 and 92 ± 18 in four groups,and median survival was 72,98,112 and 80 days,respectively.Apoptosis rate was (2.6 ± 1.1 )%,(8.6 ±3.7)%,(24.3±6.9)% and (6.8 ± 1.5)% respectively in four groups.Combination of aspirin and sorafenib significantly decreased tumor volume ( P < 0.05 ),inhibited number of lung metastases ( P < 0.01 ),and prolonged survival (P < 0.05 ) as compared with sorafenib alone group.Increased apoptosis of tumor cells (P <0.05 ) was observed by combination of aspirin and sorafenib.Nuclear factor-κB (NF-κB) was upregulated and IκBα was downregulated by sorafenib,which was reversed by combination of aspirin with sorafenib.Conclusion Aspirin significantly promoted apoptosis of tumor cells in combination with sorafenib through inhibiting sorafenib-induced NF-κB activation.%目的 观察合用阿司匹林(Aspirin)与索拉非尼(Sorafenib)对肝癌生长转移的抑制作用和促凋亡作用并探讨其机制.方法 采用人肝癌裸鼠原位模型LCI-D20,分为对照组、Sorafenib 单用组(30 mg/kg灌胃,1d1次)、Sorafenib+ Aspirin合用组、Aspirin单用组(30 mg/kg灌胃,1 d 1 次),治疗4周后观

  8. 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...

  9. Ten years of transition and... / Rika Ishii, Nick Sten

    Index Scriptorium Estoniae

    Ishii, Rika

    1999-01-01

    Ülevaade majandusreformidest Baltimaades ja Baltimaade majandussuhetest Euroopa Liidu ning Venemaaga. Tabel: SKP, inflatsioon, väliskaubandus ja välisinvesteeringud Eestis, Lätis, Leedus ja Kesk- ja Ida-Euroopa riikides aastatel 1991-1998

  10. Photocatalytic property of nitrogen and nicked codoped titanium oxides

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Yong; Jun, Kyeong Mun; Kwon, Ki Young [Dept. of Chemistry, Research Institute of Natural Science, Gyeongsang National University, Jinju (Korea, Republic of); Ahn, Seok Hoon; Hwang, Jun Yeon [Institute of Advanced Composite Materials, Korea Institute of Science and Technology, Wanju (Korea, Republic of)

    2016-11-15

    Nitrogen or nitrogen and nickel codoped TiO{sub 2}s are prepared by a hydrothermal method. The doped TiO{sub 2}s are applied to the photodecomposition of methylene blue (MB) under visible-light irradiation. The chemical and physical properties of catalysts are characterized by X-ray diffraction, transmission electron microscope, energy dispersive spectroscopy mapping, zeta potential, and Brunauer–Emmett–Teller. We found that nickel and nitrogen atoms are well dispersed in TiO{sub 2}. While the adsorption of MB on the TiO{sub 2} surface are improved by the nitrogen doping, the photodecomposition capability of MB is barely affected by the nitrogen doping level. Particularly, the photodegradation power of the nickel and nitrogen codoped TiO{sub 2} is better than that of commercial TiO{sub 2} (Degussa P25)

  11. Ten years of transition and... / Rika Ishii, Nick Sten

    Index Scriptorium Estoniae

    Ishii, Rika

    1999-01-01

    Ülevaade majandusreformidest Baltimaades ja Baltimaade majandussuhetest Euroopa Liidu ning Venemaaga. Tabel: SKP, inflatsioon, väliskaubandus ja välisinvesteeringud Eestis, Lätis, Leedus ja Kesk- ja Ida-Euroopa riikides aastatel 1991-1998

  12. Greens protest at U. S. Embassy / Nick Coleman

    Index Scriptorium Estoniae

    Coleman, Nick

    2000-01-01

    Protestijad kogunesid USA Riia saatkonna hoone ette. Väljendati oma rahulolematust USA tahtmatuse suhtes vähendada süsinikdioksüüdi paiskamist õhkkonda, mis ilmnes eelmisel kuul peetud kliimamuutuse konverentsil Haagis

  13. In the Nick of Time: Proactive Prevention of Obligation Violations

    DEFF Research Database (Denmark)

    Basin, David; Debois, Søren; Hildebrandt, Thomas

    2016-01-01

    We present a system model, an enforcement mechanism, and a policy language for the proactive enforcement of timed provisions and obligations. Our approach improves upon existing formalisms in two ways: (1) we exploit the target system's existing functionality to avert policy violations proactively...

  14. Apoptosis-Inducing Factor Participation in Bovine Macrophage Mycobacterium bovis-Induced Caspase-Independent Cell Death▿

    Science.gov (United States)

    Vega-Manriquez, X.; López-Vidal, Y.; Moran, J.; Adams, L. G.; Gutiérrez-Pabello, J. A.

    2007-01-01

    Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Previously, our group demonstrated macrophage apoptosis as a consequence of Mycobacterium bovis infection. In this study, we aimed to identify the contribution of apoptotic effector elements in M. bovis-induced CD. Bovine macrophages were either infected with M. bovis (multiplicity of infection, 10:1) or treated with an M. bovis cell extract (CFE). Structural changes compatible with CD were evaluated. Chromatin condensation was increased three times by the CFE. On the other hand, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay demonstrated that levels of DNA fragmentation induced by M. bovis and CFE were 53.7% ± 24% and 38.9% ± 14%, respectively, whereas control cells had a basal proportion of 8.9% ± 4.1%. Rates of DNA fragmentation were unaffected by the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (z-VAD). Cells treated with 100 μg of CFE for 12 h had a fivefold decrease in the level of mitochondrial outer membrane permeabilization compared to that of untreated cells. Neither M. bovis infection nor CFE treatment induced activation of caspase 3, 8, or 9. Translocation of apoptosis-inducing factor (AIF) to the nucleus was identified in 32% ± 3.5% and 26.3% ± 4.9% of M. bovis-infected and CFE-treated cells, respectively. Incubation of macrophages with z-VAD prior to infection did not alter the percentage of cells showing AIF translocation. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with AIF participation. PMID:17158896

  15. Hyperthermia sensitizes Rhizopus oryzae to posaconazole and itraconazole action through apoptosis.

    Science.gov (United States)

    Shirazi, Fazal; Pontikos, Michael A; Walsh, Thomas J; Albert, Nathaniel; Lewis, Russell E; Kontoyiannis, Dimitrios P

    2013-09-01

    The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 μg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 μg/ml) and 42°C (0.25 to 1 μg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4',6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.

  16. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality

    OpenAIRE

    TAKEDA, Kumiko; UCHIYAMA, Kyoko; KINUKAWA, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was d...

  17. A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

    OpenAIRE

    Domínguez, Fernándo; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier

    2003-01-01

    The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl trans...

  18. To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

    OpenAIRE

    2014-01-01

    Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, ...

  19. Gedichte aus den Jahren 1968 bis 1975 / Viivi Luik ; tõlk. Gisbert Jänicke

    Index Scriptorium Estoniae

    Luik, Viivi, 1946-

    1999-01-01

    Tekst saksa ja eesti keeles. Sisu: Weite = Avarus ; "Wie der Krieg..." = "Otsekui sõda..." ; "es vergehn..." = "hävivad..." ; Das Geheimnis = Saladus ; "Ob auch ich weiss..." = "Kas minagi tean..." ; "Ich redete einmal von Feldern..." = "Rääkisin väljadest ükskord..." ; Andere = Teine ; Der Tag = Päev

  20. Bulky guanidinato nickel(I) complexes: synthesis, characterization, isomerization, and reactivity studies.

    Science.gov (United States)

    Jones, Cameron; Schulten, Christian; Fohlmeister, Lea; Stasch, Andreas; Murray, Keith S; Moubaraki, Boujemaa; Kohl, Stuart; Ertem, Mehmed Z; Gagliardi, Laura; Cramer, Christopher J

    2011-01-24

    Reactions of lithium complexes of the bulky guanidinates [{(Dip)N}(2)CNR(2)](-) (Dip=C(6)H(3)iPr(2)-2,6; R=C(6)H(11) (Giso(-)) or iPr (Priso(-)), with NiBr(2) have afforded the nickel(II) complexes [{Ni(L)(μ-Br)}(2)] (L=Giso(-) or Priso(-)), the latter of which was crystallographically characterized. Reduction of [{Ni(Priso)(μ-Br)}(2)] with elemental potassium in benzene or toluene afforded the diamagnetic species [{Ni(Priso)}(2)(μ-C(6)H(5)R)] (R=H or Me), which were shown, by X-ray crystallographic studies, to possess nonplanar bridging arene ligands that are partially reduced. A similar reduction of [{Ni(Priso)(μ-Br)}(2)] in cyclohexane yielded a mixture of the isomeric complexes [{Ni(μ-κ(1)-N-,η(2)-Dip-Priso)}(2)] and [{Ni(μ-κ(2)-N,N'-Priso)}(2)], both of which were structurally characterized. These complexes were also formed through arene elimination processes if [{Ni(Priso)}(2)(μ-C(6)H(5)R)] (R=H or Me) were dissolved in hexane. In that solvent, diamagnetic [{Ni(μ-κ(1)-N-,η(2)-Dip-Priso)}(2)] was found to slowly convert to paramagnetic [{Ni(μ-κ(2)-N,N'-Priso)}(2)], suggesting that the latter is the thermodynamic isomer. Computational analysis of a model of [{Ni(μ-κ(2)-N,N'-Priso)}(2)] showed it to have a Ni-Ni bond that has a multiconfigurational electronic structure. An analogous copper(I) complex [{Cu(μ-κ(2)-N,N'-Giso)}(2)] was prepared, structurally authenticated, and found, by a theoretical study, to have a negligible Cu···Cu bonding interaction. The reactivity of [{Ni(Priso)}(2)(μ-C(6)H(5)Me)] and [{Ni(μ-κ(2)-N,N'-Priso)}(2)] towards a range of small molecules was examined and this gave rise to diamagnetic complexes [{Ni(Priso)(μ-CO)}(2)] and [{Ni(Priso)(μ-N(3))}(2)]. Taken as a whole, this study highlights similarities between bulky guanidinate ligands and the β-diketiminate ligand class, but shows the former to have greater coordinative flexibility.

  1. "Esst estnische Pasteten in estnischen Nischen" : Ilmar Laabans poetische Welt / Gisbert Jänicke

    Index Scriptorium Estoniae

    Jänicke, Gisbert, 1937-

    2000-01-01

    Artikkel sisaldab I. Laabani luuletusi saksa k.: Das Ende der Ankerkette ist des Gesanges Beginn ; Ich liebe euch ; "Die Quelle des Tods in sauberen Wolken..." ; Vögel des Selbstmords ; "Ich zerstreue mich über meinen Tisch..." ; Pornografisch ; Die Moral des Nordlichts ; Mikrokosmos 1, 20, 23, 26, 29, 30, 34, 49. Samuti sisaldab artikkel I. Laabani rootsik. luuletuse "Hur står det till?" saksak. teksti ja I. Laabani saksa k. kirjutatud luuletuse "Funkenspielstück".

  2. Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

    Energy Technology Data Exchange (ETDEWEB)

    Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.; Eakes, Thomas C.; Eto, Karina Yui; Kwong, Stephen M.; Ramsay, Joshua P.; Firth, Neville; Redinbo, Matthew R.; Christie, P. J.

    2016-01-04

    Antimicrobial resistance inStaphylococcus aureuspresents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at theoriTregion of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES proteinin vitro. Second, pSK156 and pCA347 are nonconjugativeStaphylococcus aureusplasmids that contain sequences similar to theoriTregion of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate theoriTsequences of these nonconjugative plasmidsin vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognateoriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variantoriTmimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-likeoriT. These data indicate that the conjugative relaxase intransmechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids.

    IMPORTANCEUnderstanding the mechanism of antimicrobial resistance transfer in bacteria such asStaphylococcus aureusis an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of theStaphylococcus aureusmultiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variantoriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase intransmechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

  3. Im Wind / Mari Vallisoo ; tõlk. Gisbert Jänicke

    Index Scriptorium Estoniae

    Vallisoo, Mari, 1950-2013

    2003-01-01

    Sisu: Im Wind = Tuulega ; Zwei Frauen = Kaks naist ; In Gedanken an Aleksander Levin = aleksander Levinile mõteldes ; Jetzt im Dunkel = Nüüd pimedas ; Fragen = Küsimused ; Der Zwillingsbruder = Kaksikvend

  4. Zehn Oden / Kristjan Jaak Peterson ; [Deutsch von Gisbert Jänicke

    Index Scriptorium Estoniae

    Peterson, Kristian Jaak, 1801-1822

    2001-01-01

    On ilmunud ka kogumikus "Iaak. Kristian Jaak Peterson 200" (Tln. : Eesti Keele Sihtasutus, 2001, lk. 105-122). Sisu: Der Mond = Kuu ; Freundschaft = Sõprus ; Der Sänger = Laulja ; An Gott = Jumalale ; Hoffnung = Lootus ; Ich muss Trinken. Liedchen = Ma pean jooma. Lauluke ; Ich bin wieder glücklich = Olen jälle õnnis ; Der Mensch = Inimene ; Sonnenuntergang = Päeva loojaminemine ; Herbst = Sügise.

  5. Dynamic Sheaths, in the Nick of Time or Past Their Prime?

    Science.gov (United States)

    Krajcer, Zvonimir; Parekh, Dhaval

    2016-12-01

    Risk factors for vascular complications include a sheath to femoral artery ratio (SFAR) > 1.05, use of sheaths >19 Fr, peripheral artery disease, female gender, and a learning curve. High rates of technical and procedural success were achieved with low rates of major vascular and bleeding complications. However, SFAR > 1.05, gender, diabetes, renal insufficiency, calcification, tortuosity, or aneurysm were not independent risk factors in this study. Despite an SFAR > 1.05 in 23 patients, increased rates of vascular complications were not seen using the SoloPath sheath. These findings may increase the pool of transfemoral TAVI patients.

  6. Zehn Oden = Kümme oodi / Kristjan Jaak Peterson ; Deutsch von Gisbert Jänicke

    Index Scriptorium Estoniae

    Peterson, Kristjan Jaak, 1801-1822

    2001-01-01

    Sisu: An Gott ; Sonnenuntergang ; Herbst ; Der Mensch ; Der Mond ; Freundschaft ; Ich muss trinken. Liedchen ; Hoffnung ; Ich bin wieder glücklich ; Der Sänger. Orig.: Jummalale ; Päva loja-minneminne ; Süggise ; Innimenne ; Kuu ; Söbradus ; Ma pean joma. Lauloke ; Lotus ; Ollen jälle önnis ; Laulja

  7. The Neglected Protagonist:The Functions of the Role Nick Carraway in The Great Gatsby

    Institute of Scientific and Technical Information of China (English)

    李鹏飞

    2013-01-01

    In F. Scott Fitzgerald’s world famous novel The Great Gatsby, Nicolas“Nick”Carraway is not only a first-person nar-rator, but also a convincing narrator, an emotion depositor, and a structure organizer. He is the figure of equal weight with Jay Gatsby and Daisy Buchanan, the two conceived protagonists in this book.

  8. Venemaa näitab Ida-Euroopas jõuliselt muskleid / Nick Thorpe

    Index Scriptorium Estoniae

    Thorpe, Nick

    2007-01-01

    Euroopa Komisjon on väljendanud muret Venemaa plaanide pärast omandada gaasi- ja naftajuhtmeid ning muid strateegilisi varasid, samal ajal ostavad venelased kokku energeetika- ja muidki firmasid eelkõige kunagi NSV Liidu mõjualas olnud riikides

  9. Nick Hales Award Lecture 2011: glucocorticoids and early life programming of cardiometabolic disease.

    Science.gov (United States)

    Reynolds, R M

    2012-10-01

    Epidemiological studies have demonstrated an association between low birthweight and a range of diseases in adult life including cardiometabolic and psychiatric diseases. One of the key mechanisms proposed to underlie early life 'programming' of disease is overexposure of the developing foetus to glucocorticoids. This review will explore the data from human studies that glucocorticoids are not only mediators of programming, but also targets of programming. Cohort studies of men and women of known birthweight have demonstrated that low birthweight is associated with high fasting cortisol levels. In healthy individuals and in people with type 2 diabetes who are at high cardiovascular risk, there is a similar association between high fasting cortisol and the metabolic syndrome. The high cortisol levels appear to be due to activation of the hypothalamic-pituitary-adrenal (HPA) axis though detailed studies to further explore central negative feedback sensitivity are required. The evidence in humans that glucocorticoids mediate programming is more scanty, though changes in maternal body composition, stress and anxiety levels and activity of the placental barrier enzyme 11 β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) may all influence maternal HPA axis activity. Emerging studies are supportive that high maternal cortisol levels in humans and/or deficiencies placental 11β-HSD2 humans are associated with lower birthweight and adverse metabolic and neurocognitive outcomes in the offspring.

  10. Výzkum diváka Strašnického divadla

    OpenAIRE

    Humlová, Jana

    2012-01-01

    This bachelor thesis focuses on marketing applied by theatres. Firstly there is a definition of marketing in general and then the thesis focuses on differences in marketing applied by cultural organizations, especially by theatres. Then it shows diverse types of theatres. Afterwards the thesis becomes less theoretical and gets more concrete and it explains the term marketing mix on example of Strašnice theatre. The practical part is represented by a marketing research. The purpose of the rese...

  11. Gedichte / [Konnula, Margus] Contra ; aus dem Estnischen von Gisbert Jänicke

    Index Scriptorium Estoniae

    Contra, pseud., 1974-

    2002-01-01

    Sisu : Die Nacht auf dem Busbahnhof = Öö bussijaamas ; Ich bin schwarz und schmutzig = Olen must ja räpane ; "wir sind da auf dem bild..." = "oleme siin pildi peal..." ; Lebensgrundlage = Elu alus ; Der Volksvertreter = Rahvaasemik ; Das Messer hinterm Rücken in der Hand = Selja taga nuga käes ; Wo = Kus ; Gute Nachbarn = Head naabrid

  12. Tabellae et tabulata : [luuletused] / Hasso Krull ; aus dem Estnischen von Gisbert Jänicke

    Index Scriptorium Estoniae

    Krull, Hasso, 1964-

    2001-01-01

    Sisu : Der Kopf dreht sich [Pea käib ringi] ; Gedicht [Luuletus] ; Vier Tafeln 1-4 [Neli tabelit 1-4] ; I : "Gerade hier entlang kamen wir in den Verkehr..." [I : "Just siitkaudu tulime liikumisele lähedale..."] ; II : "Angenehmer Kaffeeduft. Von einer Stärke, die..." [II : "Meeldiv kohvilõhn. Sellel on jõudu, mis toob..."] ; III : "Hesekiel schreibt, "ihre Felgen waren so hoch, dass ich..." [III : "Hesekiel märgib, et "nende pöiad olid kõrged ning..."] ; IV : "Zwangsläufig wird der Luftdruck immer spürbarer..." [IV : "Paratamatult muutub õhurõhk üha märgatavamaks ..."] ; V : "Ich war im Wald, sammelte Blätter von den Bäumen, ass sie..." [V : "Olin metsa all, korjasin puulehti, sõin neid..."] ; VI : "Der Grund für die Gewalt ist immer der andere. Der..." ["Vägivalla alus on just üks Teine. Kes..."] ; - : "Grüsst euch. Euch auch..." [-: "No tere. Tere ka..."

  13. Tabellae et tabulata : [luuletused] / Hasso Krull ; aus dem Estnischen von Gisbert Jänicke

    Index Scriptorium Estoniae

    Krull, Hasso, 1964-

    2001-01-01

    Sisu : Der Kopf dreht sich [Pea käib ringi] ; Gedicht [Luuletus] ; Vier Tafeln 1-4 [Neli tabelit 1-4] ; I : "Gerade hier entlang kamen wir in den Verkehr..." [I : "Just siitkaudu tulime liikumisele lähedale..."] ; II : "Angenehmer Kaffeeduft. Von einer Stärke, die..." [II : "Meeldiv kohvilõhn. Sellel on jõudu, mis toob..."] ; III : "Hesekiel schreibt, "ihre Felgen waren so hoch, dass ich..." [III : "Hesekiel märgib, et "nende pöiad olid kõrged ning..."] ; IV : "Zwangsläufig wird der Luftdruck immer spürbarer..." [IV : "Paratamatult muutub õhurõhk üha märgatavamaks ..."] ; V : "Ich war im Wald, sammelte Blätter von den Bäumen, ass sie..." [V : "Olin metsa all, korjasin puulehti, sõin neid..."] ; VI : "Der Grund für die Gewalt ist immer der andere. Der..." ["Vägivalla alus on just üks Teine. Kes..."] ; - : "Grüsst euch. Euch auch..." [-: "No tere. Tere ka..."

  14. Venemaa näitab Ida-Euroopas jõuliselt muskleid / Nick Thorpe

    Index Scriptorium Estoniae

    Thorpe, Nick

    2007-01-01

    Euroopa Komisjon on väljendanud muret Venemaa plaanide pärast omandada gaasi- ja naftajuhtmeid ning muid strateegilisi varasid, samal ajal ostavad venelased kokku energeetika- ja muidki firmasid eelkõige kunagi NSV Liidu mõjualas olnud riikides

  15. "Esst estnische Pasteten in estnischen Nischen" : Ilmar Laabans poetische Welt / Gisbert Jänicke

    Index Scriptorium Estoniae

    Jänicke, Gisbert, 1937-

    2000-01-01

    Artikkel sisaldab I. Laabani luuletusi saksa k.: Das Ende der Ankerkette ist des Gesanges Beginn ; Ich liebe euch ; "Die Quelle des Tods in sauberen Wolken..." ; Vögel des Selbstmords ; "Ich zerstreue mich über meinen Tisch..." ; Pornografisch ; Die Moral des Nordlichts ; Mikrokosmos 1, 20, 23, 26, 29, 30, 34, 49. Samuti sisaldab artikkel I. Laabani rootsik. luuletuse "Hur står det till?" saksak. teksti ja I. Laabani saksa k. kirjutatud luuletuse "Funkenspielstück".

  16. Im Wind / Mari Vallisoo ; tõlk. Gisbert Jänicke

    Index Scriptorium Estoniae

    Vallisoo, Mari, 1950-2013

    2003-01-01

    Sisu: Im Wind = Tuulega ; Zwei Frauen = Kaks naist ; In Gedanken an Aleksander Levin = aleksander Levinile mõteldes ; Jetzt im Dunkel = Nüüd pimedas ; Fragen = Küsimused ; Der Zwillingsbruder = Kaksikvend

  17. An improved method for the synthesis of mercurated dUTP. Enzymic synthesis of Hg-labelled DNA of high molecular weight suitable for use in an image based DNA sequencing strategy.

    Science.gov (United States)

    Bridgman, A J; Petersen, G B

    1996-01-01

    The development of high-resolution scanning-probe microscopes has reawakened interest in the possibility of sequencing large nucleic acid molecules by direct imaging. Such an approach would be facilitated by the availability of effective methods for increasing contrast by labelling specific nucleotides, and the utility of introducing mercury atoms into complete DNA molecules through the enzymic polymerisation of mercurated pyrimidine deoxynucleoside triphosphates has been re-investigated. A simplified and improved method for the synthesis of a heat- and thiol-stable, mercurated derivative of deoxyuridine triphosphate in high yield and the incorporation of this precursor into full-length copies of a single-stranded phage M13 template are described. The DNA product has been fully characterised and the quantitative and specific replacement of thymidylic acid residues by the mercurated analogue demonstrated.

  18. Continuous lactation effects on mammary remodeling during late gestation and lactation in dairy goats

    DEFF Research Database (Denmark)

    Safayi, Sina; Theil, Peter Kappel; Hou, Lei

    2010-01-01

    dropped to below 50 g/d. Mammary biopsies were obtained from each udder half just before the NL gland was dried off (before dry period), within the first 2 wk after drying-off (early dry period, samples available only for NL glands), in the mid dry period, within the last 2 wk before parturition (late dry...... period), and at d 1 (the day of parturition), 3, 10, 60, and 180 of lactation. Mammary morphology was characterized in biopsies by quantitative histology, and cell turnover was determined by immunohistochemistry (terminal deoxynucleotidyl transferase dUTP nick end labeling and Ki-67). Transcription...

  19. Ce4+-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    葛志强; 元英进; 王艳东; 马振毅; 胡宗定

    2002-01-01

    The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged "DNA ladder" on agarose gel electrophoresis. TdT-mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′-OH termini. These results suggest that Ce4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product-Taxol.

  20. Nuclear DNA fragmentation during cell death of short-lived ray tracheids in the conifer Pinus densiflora.

    Science.gov (United States)

    Nakaba, Satoshi; Kubo, Takafumi; Funada, Ryo

    2011-05-01

    One key event in the programmed cell death is nuclear DNA fragmentation. We investigated the timing of nuclear DNA fragmentation during the cell death of short-lived ray tracheids in Pinus densiflora using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Fluorescence due to TUNEL was detected only in deformed nuclei that lacked obvious chromatin in ray tracheids that were adjacent to ray tracheids that no longer contained nuclei. Our observations revealed that nuclear DNA fragmentation occurred only at the final stage of cell death in ray tracheids in situ.

  1. Sperm DNA integrity in relation to exposure to environmental perfluoroalkyl substances - a study of spouses of pregnant women in three geographical regions

    DEFF Research Database (Denmark)

    Specht, Ina Olmer; Hougaard, Karin; Spanò, Marcello;

    2012-01-01

    S) and concurrent DNA damage in spermatozoa by sperm chromatin structure assay (SCSA) and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, apoptotic markers in semen (Fas-receptor and Bcl-xL), and reproductive hormones in serum. No association between PFASs and SCSA, apoptotic...... markers or reproductive hormones emerged. We observed a slight increase in SHBG and TUNEL-positivity with increased PFOA exposure in men from Greenland. Thus, consistent evidence that PFAS exposure interferes with sperm DNA fragmentation, apoptosis or reproductive hormones was not found....

  2. TUNEL法计数体外培养细胞凋亡细胞数的体会

    Institute of Scientific and Technical Information of China (English)

    张丽杰; 赵振军; 易小兵; 余小平

    2002-01-01

    @@ TUNEL是末端脱氧核苷酸转移酶(terminaldeoxynucleotidyltransferase)介导的dUTP原位切口末端标记(terminal deoxynucle-otidyltransferase dUTP nick end labeling,TUNEL)技术,是原位检测细胞凋亡情况的一种基本方法,目前应用很广泛.作者在顺铂诱导人卵巢癌细胞系HO-8910细胞凋亡的研究中,使用到了该技术,现将使用过程中的体会报告如下.

  3. Safety evaluation of poly(lactic-co-glycolic acid)/poly(lactic-acid) microspheres through intravitreal injection in rabbits.

    Science.gov (United States)

    Rong, Xianfang; Yuan, Weien; Lu, Yi; Mo, Xiaofen

    2014-01-01

    Poly(lactic-co-glycolic acid) (PLGA) and/or poly(lactic-acid) (PLA) microspheres are important drug delivery systems. This study investigated eye biocompatibility and safety of PLGA/PLA microspheres through intravitreal injection in rabbits. Normal New Zealand rabbits were randomly selected and received intravitreal administration of different doses (low, medium, or high) of PLGA/PLA microspheres and erythropoietin-loaded PLGA/PLA microspheres. The animals were clinically examined and sacrificed at 1, 2, 4, 8, and 12 weeks postadministration, and retinal tissues were prepared for analysis. Retinal reactions to the microspheres were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end staining and glial fibrillary acidic protein immunohistochemistry. Retinal structure changes were assessed by hematoxylin and eosin staining and transmission electron microscopy. Finally, retinal function influences were explored by the electroretinography test. Terminal deoxynucleotidyl transferase-mediated dUTP nick end staining revealed no apoptotic cells in the injected retinas; immunohistochemistry did not detect any increased glial fibrillary acidic protein expression. Hematoxylin and eosin staining and transmission electron microscopy revealed no micro- or ultrastructure changes in the retinas at different time points postintravitreal injection. The electroretinography test showed no significant influence of scotopic or photopic amplitudes. The results demonstrated that PLGA/PLA microspheres did not cause retinal histological changes or functional damage and were biocompatible and safe enough for intravitreal injection in rabbits for controlled drug delivery.

  4. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  5. Gedichte : [aus "Talv"¡ ("Winter")] : [luuletused] / Hasso Krull ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Krull, Hasso, 1964-

    2006-01-01

    Sisu: Winter : "Die Demokratie. Sie ist in gewissem Sinn ein Kastensystem..." ; "Die Erdmännchen. Ihre Hochzeit..." ; "Die Bären. Wachen auf und fragen..." ; "Die Wanzen. Wohnten in der Matratze..." ; "Die Folter. Schon die christlich Inquisition..." ; "Das Gedächtnis. Alle schreiben sie davon..." ; "Einen Gott gibt es...". Orig.: Talv : "Demokraatia. See on omamoodi kastisüsteem..." ; "Kümnekingalised. Nende pulm..." ; "Karud. Ärkavad üles ja küsivad..." ; "Lutikad. Elasid madratsi sees..." ; "Piinamine. Juba kristlik inkvisitsioon..." ; "Mälu. Kõik kirjutavad sellest..." ; "Üks jumal on olemas..."

  6. Gedichte : [aus "Talv"¡ ("Winter")] : [luuletused] / Hasso Krull ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Krull, Hasso, 1964-

    2006-01-01

    Sisu: Winter : "Die Demokratie. Sie ist in gewissem Sinn ein Kastensystem..." ; "Die Erdmännchen. Ihre Hochzeit..." ; "Die Bären. Wachen auf und fragen..." ; "Die Wanzen. Wohnten in der Matratze..." ; "Die Folter. Schon die christlich Inquisition..." ; "Das Gedächtnis. Alle schreiben sie davon..." ; "Einen Gott gibt es...". Orig.: Talv : "Demokraatia. See on omamoodi kastisüsteem..." ; "Kümnekingalised. Nende pulm..." ; "Karud. Ärkavad üles ja küsivad..." ; "Lutikad. Elasid madratsi sees..." ; "Piinamine. Juba kristlik inkvisitsioon..." ; "Mälu. Kõik kirjutavad sellest..." ; "Üks jumal on olemas..."

  7. Uroboros : [aus "Uroboros"] : [luuletused] / Ene Mihkelson ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Mihkelson, Ene, 1944-

    2006-01-01

    Sisu: Uroboros : "In der Nacht fragt heimlich jemand Wie heisst du..." ; "Am Meer stand ein Mann mit einer Sense..." ; "An dem Tag als es Kugeln regnete..." ; "Zum Zeitvertrieb ersteigerte ich mir auf einer Auktion ein paar..." ; "Sie hat sich mit der Zeit eingelassen und daraus..." ; Das Feld ; "Mutter ist wieder bei mir obgleich..." ; "Wir waren ein Mädchen Wir redeten..." ; "Als ich vier Jahre alt wurde Da wurde so viel..." ; "Wer ist dein Vater Der Fremde aus der Ferne..." ; "Das letzte Wort Das letzte Das letzte Das allein...". Orig.: Uroboros : "Öö aegu keegi küsib sala Mis su nimi on..." ; "Mere ees seisis vikatiga mees..." ; "Sel päeval kui sadas kuulivihma..." ; "Ajaviiteks ostsin enampakkumisel sõpru..." ; "Ta ajaga läinud on sohki ja sellest..." ; Põld ; "Mu ema on jälle mu juures kuigi..." ; "Meid oli üks türduk Me rääkisime..." ; "Kui olin saanud neljaseks Nii väga kiitis..." ; "Kes on su isa Kaugelt tulnud võõras..." ; "Viimast sõna Viimast Viimast Ainu..."

  8. Der Meteorologe : (aus dem Band "V". Tallinn 1998) / Elo Viiding ; aus dem Estnischen von Gisbert Jänicke

    Index Scriptorium Estoniae

    Viiding, Elo, 1974-

    2002-01-01

    Sisu : Die Möglichkeit des Meteorologen = Meteoroloogi võimalikkusest ; "Der Meteorologe kam 1990 in die Stadt..." = "Meteoroloog saabus linna aastal 1990..." ; "Was wäre dir "Arbeit" des Meteorologen..." = "Mis oleks meteoroloogi töö..." ; "Und ein Unglück für den Meteorologen ist es auch..." = "Ja Meteoroloogi õnnetus on veel see..." ; Angst vor dem Altwerden des Meteorologen = Hirm Meteoroloogi vanakssaamise ees ; Fest. Geschenk = Pidu. Kink ; "Wenn der Meteorologe eine Grösse sieht, ist er darüber..." = "Kui meteoroloog näeb suurust, on ta selle kohal..." ; Der Meteorologe wird im Saal erwartet = Meteoroloogi oodatakse saali ; "Das Abkommen mit der Meteorologenerwartung kündigen..." = "Katkestada leping meteoroloogiootusega..." ; "Die "Wege des Herrn" sind der Meteorologe..." = "Looja tee" on Meteoroloog..." ; Von dem Fremden, der im Saal den Meteorologen traf = Võõra lugu, kes Meteoroloogi saalis kohtas ; "Den Fremden hervorzuhusten, der von dem..." = "Köhida enesest välja võõras, kes tahtis teha..." ; Der Fremde beruhigt sich nicht = Võõras ei jää rahule

  9. Uroboros : [aus "Uroboros"] : [luuletused] / Ene Mihkelson ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Mihkelson, Ene, 1944-2017-

    2006-01-01

    Sisu: Uroboros : "In der Nacht fragt heimlich jemand Wie heisst du..." ; "Am Meer stand ein Mann mit einer Sense..." ; "An dem Tag als es Kugeln regnete..." ; "Zum Zeitvertrieb ersteigerte ich mir auf einer Auktion ein paar..." ; "Sie hat sich mit der Zeit eingelassen und daraus..." ; Das Feld ; "Mutter ist wieder bei mir obgleich..." ; "Wir waren ein Mädchen Wir redeten..." ; "Als ich vier Jahre alt wurde Da wurde so viel..." ; "Wer ist dein Vater Der Fremde aus der Ferne..." ; "Das letzte Wort Das letzte Das letzte Das allein...". Orig.: Uroboros : "Öö aegu keegi küsib sala Mis su nimi on..." ; "Mere ees seisis vikatiga mees..." ; "Sel päeval kui sadas kuulivihma..." ; "Ajaviiteks ostsin enampakkumisel sõpru..." ; "Ta ajaga läinud on sohki ja sellest..." ; Põld ; "Mu ema on jälle mu juures kuigi..." ; "Meid oli üks türduk Me rääkisime..." ; "Kui olin saanud neljaseks Nii väga kiitis..." ; "Kes on su isa Kaugelt tulnud võõras..." ; "Viimast sõna Viimast Viimast Ainu..."

  10. 唯一不变的是信息本身——专访EMC公NCTO Jeffry Nick

    Institute of Scientific and Technical Information of China (English)

    欧阳璟

    2007-01-01

    1979年,Richard(Dick)Egan和Roger Marino共同创建了一家伟大的IT公司,经过28年时间,它已经发展成为一个拥有31000多名员工,年收入超过111亿美元的IT巨头。两位创始人的姓氏被刻在公司的名字上,这就是EMC。今天,我们看到这家公司的名字已经成为EMC2,这非常像一个经典的物理学公式:E=mc2。这个质能转换公式的背后代表着一些永恒不变的规律,就像EMC一贯坚持的那样,因为他们相信在IT世界,唯一不变的是信息本身。

  11. Effects of propofol on neuronal apoptosis and aquaporin-4 expression in a rat model of traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    Jianfang Song; Xiangyu Ji; Zangong Zhou

    2008-01-01

    BACKGROUND: Several studies have demonstrated that propofol exhibits protective effects in the central nervous system. OBJECTIVE: To observe the effects of propofol on neuronal apoptosis and aquaporin-4 (AQP-4) expression in a rat model of traumatic brain injury and to further investigate the mechanisms of action. DESIGN, TIME AND SETTING: The present neuronal, pathomorphological experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical College between April 2007 and March 2008. MATERIALS: Traumatic brain injury was induced by free falling objects in 150 healthy, male, Wistar rats. Propotol was produced by AstraZeaeca, China. Rabbit anti-rat AQP-4 polyclonal antibody, SABC inununohistochemistry kit, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) kit were purchased from Wuhan Boster Bioengineering Co., Ltd., China. METHODS: All 150 rats were randomly and evenly divided into lesion-only and propofol-treated groups. One hour after traumatic brain injury, propofol-treated animals received 1% propofol (10 mg/kg) through the caudal vein, followed by a sustained perfusion of 30 mg/kg propofol per hour for 2 hours, while the lesion-only group received equal volumes of physiological saline in parallel. MAIN OUTCOME MEASURES: At 6, 12, 24, 48, and 72 hours after traumatic brain injury, morphological changes in the peritraumatic and adjacent brain areas were analyzed in all rats by hematoxylin-eosin (HE) staining. In addition, cellular apoptosis was detected by TUNEL assay and the number of AQP-4-positive cells was determined by immunohistochemistry techniques. Brain water content was calculated as the ratio of dry to wet tissue weight. RESULTS: HE staining results demonstrated that, in the lesion-only group, the peritraumatic area exhibited neuronal and glial cell necrosis and disintegration. The adjacent area displayed swollen neuronal perikarya and vascular endothelial cells, cellular edema

  12. Protective effects of berberine against amyloid beta-induced toxicity in cultured rat cortical neurons

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Yanjun Zhang; Shuai Du; Mixia Zhang

    2011-01-01

    Berberine, a major constituent of Coptidis rhizoma, exhibits neural protective effects. The present study analyzed the potential protective effect of berberine against amyloid G-induced cytotoxicity in rat cerebral cortical neurons. Alzheimer's disease cell models were treated with 0.5 and 2 μmol/Lberberine for 36 hours to inhibit amyloid G-induced toxicity. Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons. In addition, western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons, which coincided with significantly decreased abnormal up-regulation of activated caspase-3. These results showed that berberine exhibited a protective effect against amyloid 13-induced cytotoxicity in cultured rat cortical neurons.

  13. Successful ram semen cryopreservation with lyophilized egg yolk-based extender.

    Science.gov (United States)

    Alcay, Selim; Berk Toker, M; Gokce, Elif; Ustuner, Burcu; Tekin Onder, N; Sagirkaya, Hakan; Nur, Zekariya; Kemal Soylu, M

    2015-10-01

    The aim of this study was to evaluate the effects of lyophilized egg yolk extender on ram semen cryopreservation. Ejaculates with a thick consistency, rapid wave motion (3-5 on a 0-5 scale) and >75% initial motility were pooled. Sperm were diluted to final concentration of 1/5 (semen/extender) in lyophilized egg yolk or fresh egg yolk extenders using two-step dilution method. The equilibrated semen was frozen in 0.25 mL straws. Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) at three time points: after dilution with extender A, equilibration and post-thaw. The results showed that freezing and thawing procedures (dilution, equilibration and thawing) had negative effects on motility (Pram semen.

  14. Inhibition of TYRO3/Akt signaling participates in hypoxic injury in hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Yan-zhen Zhu; Wei Wang; Na Xian; Bing Wu

    2016-01-01

    In this study, we investigated the role of the TYRO3/Akt signaling pathway in hypoxic injury to hippocampal neurons. 3-(4,5-Dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that hypoxia inhibited the proliferation and viability of hippocampal neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that hypoxia induced neuronal apoptosis in a time-dependent manner, with a greater number of apoptotic cells with longer hypoxic exposure. Immunolfuorescence labeling revealed that hypoxia suppressed TYRO3 expression. Western blot assay showed that hypoxia decreased Akt phosphorylation levels in a time-de-pendent manner. Taken together, these ifndings suggest that hypoxia inhibits the proliferation of hippocampal neurons and promotes apoptosis, and that the inhibition of the TYRO3/Akt signaling pathway plays an important role in hypoxia-induced neuronal injury.

  15. Study of apoptosis in human liver cancers

    Institute of Scientific and Technical Information of China (English)

    Chang-Min Shan; Juan Li

    2002-01-01

    AIM: To investigate the action of apoptosis in occurrence ofliver cacinornas in vivo and the biological effect of Solanumlyratum Thumb on BEL-7404 cell line inducing apoptosis invitro.METHODS: The apoptosis in the liver carcinoma wasdetected with terminal deoxynucl neotidyl transferasemediated dUTP nick end labelling (TUNEL); the cancer cellscultured in DMED medium were treated with extract ofSolanum lyratum Thumb and observed under microscope,and their DNA was assayed by gel electrophoresis.RESULTS: In vivo apoptotic cells in the cancer adjacenttissues inceased; in vitro treatment of liver cancers withextract of Solanum lyratum Thumb could induce the cells tomanifest a typical apoptotic morphology. Their DNA wasfractured and a characteristic ladder pattem could be foundusing electrophoresis.CONCLUSION: In vivo the apoptosis of carcinomas waslower; maybe the cells divided quickly and then the cancersoccurred. In the cancer adjacent tissues, the apoptosispricked up, and in vitro Solarium lyratum Thumb couldinduce the apoptosis of BEL-7404 cells.

  16. Ammonia exposure induces oxidative stress, endoplasmic reticulum stress and apoptosis in hepatopancreas of pacific white shrimp (Litopenaeus vannamei).

    Science.gov (United States)

    Liang, Zhongxiu; Liu, Rui; Zhao, Depeng; Wang, Lingling; Sun, Mingzhe; Wang, Mengqiang; Song, Linsheng

    2016-07-01

    Ammonia is one of major environmental pollutants in the aquatic system that poses a great threat to the survival of shrimp. In the present study, the mRNA expression of endoplasmic reticulum (ER) stress marker and unfolded protein response (UPR) related genes, as well as the change of redox enzyme and apoptosis were investigated in hepatopancreas of the pacific white shrimp, Litopenaeus vannamei after the exposure of 20 mg L(-1) total ammonia nitrogen (TAN). Compared with the control group, the superoxide dismutase (SOD) activity in hepatopancreas decreased significantly (p vannamei after exposure to ammonia by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The results indicated that ammonia exposure could induce oxidative stress, which further caused ER stress and apoptosis in hepatopancreas of L. vannamei.

  17. The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

    Institute of Scientific and Technical Information of China (English)

    BEN Yali; LIU Deli; ZHU Dali; ZHU Derui; LUO Qin

    2006-01-01

    The difference of sensitivity to indole- 3-acetic acid ( IAA ) combined with horseradish peroxidase (HRP) in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTT assay. The cell cycle and apoptosis of K562 and BXPC- 3 cells were examined by a fluorescence flow cytometer (FCM) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC- 3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.

  18. Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

    Institute of Scientific and Technical Information of China (English)

    Serafín Pérez-Cerezales; Alberto Miranda; Alfonso Gutiérrez-Adán

    2012-01-01

    It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin,which acquires fully condensed status at the caput epididymidis.The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay,the comet assay,the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation.Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay.

  19. Changes of Apoptosis in Rats of Acute Ischemic Renal Injury under Treatment of Tetrandrine

    Institute of Scientific and Technical Information of China (English)

    钱玲梅; 王笑云; 冷静

    2002-01-01

    ObjectiveTo elucidate the effect of tetrandrine on acute ischemic renal injury and its relation with apoptosis.MethodsA model for bilateral post-ischemic renal injury in rats was developed by clamping renal pedicles for 45 min.Renal tissular DNA fragmentation analysis and renal tissular HE staining were used.Also quantitative analysis of apoptosis in injured renal tubular epithelium was carried out by using TdT-mediated dUTP nick and labeling (TUNEL).ResultsApoptosis of renal tubular epithelium increased in acute ischemic renal injury.Tetrandrine could remarkably decrease the level of apoptosis in injured renal tubule while protecting renal tissue against the ischemic injuries.ConclusionTetrandrine could adjust the level of apoptosis in renal tubular epithelium and alleviate renal tissular injury.``

  20. Changes of Apoptosis in Rats of Acute Ischemic Renal Injury under Treatment of Tetrandrine

    Institute of Scientific and Technical Information of China (English)

    钱玲梅; 王笑云; 等

    2002-01-01

    Objective To elucidate the effect of tetrandrine on acute ischemic renal injury and its relation with apoptosis.Methods A model for bilateral post-ischemic renal injury in rats was developed by clamping renal pedicles for 45 min.Renal tissular DNA fragmentation analysis and renal tissular HE staining were used.Also quantitative analysis of apoptosis in injured renal tubular epithelium was carried out by using TdT-mediated dUTP nick and labeling(TUNEL).Results Apoptosis of renal tubular epithelium increased in acute ischemic renal injury.Tetrandrine could remarkably decrease the level of apoptosis in injured renal tubule while protecting renal tissue against the ischemic injuries.Conclusion Tetrandrine could adjust the level of apoptosis in renal tubular epithelium and alleviate renal tissular injury.

  1. Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

    Institute of Scientific and Technical Information of China (English)

    Yanan Cai; Xiaodong Yuan; Ya Ou; Yanhui Lu

    2011-01-01

    β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro. However, because of the short survival time of the differentiated cells, clinical applications for this technique are limited. As such, we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy. The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended. Taken together, these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death. However, the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

  2. TUNEL法检测体外培养细胞凋亡的体会

    Institute of Scientific and Technical Information of China (English)

    刘术娟; 赵天如; 张乃鑫

    2001-01-01

    @@  TUNEL(TdT-mediated dUTP nick end labeling,TdT介导的脱氧尿嘧啶缺口末端标记)是用于原位检测细胞凋亡情况的一种基本方法。作者在绿茶儿茶素(GTC)诱导BEL-7402人肝癌细胞凋亡的研究中,对培养于盖玻片的BEL-7402细胞(盖片培养细胞)和BEL-7402细胞涂片(培养细胞涂片)进行了TUNEL染色效果的比较。

  3. Apoptosis in Primary Oral Squamous Cell Carcinomas without Lymph Node Metastases

    Institute of Scientific and Technical Information of China (English)

    YAN Zhi; WANG Guomin; XIE Zhiyong; ZHANG Handong

    2005-01-01

    The apoptosis in primary oral squamous cell carcinomas (OSCCs) without lymph node (LN) metastases and its relation with clinical stages and pathological grades was investigated. The terminal deoxynucleotidyl trasferase (TdT)-mediated dUTP nick end labeling (TUNEL) was used to detect the apoptotic cells in 15 cases of OSCCs. The percentage of apoptotic cells among tumor cells were calculated as apoptotic index (AI). The results showed that in all 15 cases of OSCCs,apoptotic cells could be visualized by TUNEL with AI ranging from 0.03 to 0.92 (average 0.32).AI was significantly negatively correlated with pathological grades (P<0. 05). It was concluded that the apoptotic rate was related to the malignant degree of OSCCs without LN metastases.

  4. Apoptotic cell death of cerebellar granule neurons in genetically ataxia (ax) mice.

    Science.gov (United States)

    Ohgoh, M; Yamazaki, K; Ogura, H; Nishizawa, Y; Tanaka, I

    2000-07-21

    An autosomal recessive neurological mutant, ataxia (ax) mouse, was investigated to determine whether neuronal cell death occurs in the brain. The brains of homozygotes (ax(J)/ax(J)) and phenotypically normal littermates (ax(J)/+ or +/+) aged at 23-38 days were examined by the terminal dUTP nick-end-labeling (TUNEL) method. A few TUNEL-positive cells were observed in the granule cell layer of the cerebellum, the dentate gyrus, and the olfactory bulb of normal mice. In the affected mice, the number of TUNEL-positive cells was significantly increased in the cerebellum, particularly in the granule cell layer, compared to normal littermates. The findings suggest that ax mice will be useful as a model for studies on the genetic basis of apoptotic neuronal cell death.

  5. Hybrid male sterility is caused by mitochondrial DNA deletion.

    Science.gov (United States)

    Hayashida, Kenji; Kohno, Shigeru

    2009-07-01

    Although it is known that the hybrid male mouse is sterile just like any other animal's heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.

  6. Effect of malathion on the male reproductive organs of earthworms, Eisenia foetida

    Institute of Scientific and Technical Information of China (English)

    Omar Espinoza-Navarro; Eduardo Bustos-Obregón

    2005-01-01

    Aim: To observe the cytotoxic effect of the organophosphate insecticide malathion in the reproductive tissues of the earthworms, Eisenia foetida. Methods: Worms were nourished in soil treated with malathion at single sub-lethal exposure. The body weights were recorded and male reproductive organs evaluated. Results: Malathion-treated animals showed a significant reduction in body weight in a dose-dependent manner. Malathion treatment modified the disposition of spermatozoa in the basal epithelium of the spermatheca. The Br-deoxyuridine test showed a significant rise in cells in phase S on days 5 and 15. Also, a higher percentage of spermatogonia with fragmented DNA were observed by means of the TdT-mediated dUTP nick-end labeling (TUNEL) technique in the spermatheca of treated animals. Conclusion: Treatment with malathion decreased the body weight and the spermatic viability in spermatheca,altering the cell proliferation and modifying the DNA structure of spermatogonia.

  7. Trace amounts of copper induce neurotoxicity in the cholesterol-fed mice through apoptosis.

    Science.gov (United States)

    Lu, Jun; Zheng, Yuan-Lin; Wu, Dong-Mei; Sun, Dong-Xu; Shan, Qun; Fan, Shao-Hua

    2006-12-11

    Evidence has been gathered to suggest that trace amounts of copper induce neurotoxicity by interaction with elevated cholesterol in diet. Copper treatment alone showed no significant learning and memory impairments in behavioral tasks. However, copper-induced neurotoxicity was significantly increased in mice given elevated-cholesterol diet. Trace amounts of copper decreased the activity of SOD and increased the level of malondialdehyde (MDA) in the brain of cholesterol-fed mouse. Copper also caused an increase in amyloid precursor protein (APP) mRNA level and the activation of caspase-3 in the brain of cholesterol-fed mice. The apoptosis-induced nuclear DNA fragmentation was detected in the brain of those mice by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling staining. These findings suggest that trace amounts of copper induce neurotoxicity in cholesterol-fed mice through apoptosis caused by oxidative stress.

  8. Uterosacral ligament smooth muscle cell apoptosis is increased in women with uterine prolapse.

    Science.gov (United States)

    Takacs, Peter; Nassiri, Mehdi; Gualtieri, Marc; Candiotti, Keith; Medina, Carlos A

    2009-05-01

    The purpose of this study was to compare the smooth muscle content and apoptosis of the uterosacral ligament in women with and without uterine prolapse. Uterosacral ligaments were sampled in women with (n = 9) or without (n = 9) uterine prolapse undergoing hysterectomy. Smooth muscle of the uterosacral ligament was identified by immunohistochemistry. Digital image analysis was used to determine the fractional area of smooth muscle in the histologic cross sections. Apoptosis was assessed by terminal deoxynucelotidyl-transferase-mediated dUTP nick-end-labeling method. The fractional area of nonvascular smooth muscle in the uterosacral ligament of women with uterine prolapse was significantly decreased compared to women without prolapse (0.32 +/- 0.12 vs. 0.42 +/- 0.03, P = .02) and the apoptotic index was significantly higher compared to women without prolapse (0.20 +/- 0.06 vs. 0.08 +/- 0.04, P uterine prolapse compared to women without prolapse.

  9. Detection of apoptotic cells using immunohistochemistry.

    Science.gov (United States)

    Newbold, Andrea; Martin, Ben P; Cullinane, Carleen; Bots, Michael

    2014-11-03

    Immunohistochemistry is commonly used to show the presence of apoptotic cells in situ. In this protocol, B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples are fixed and sectioned, and fragmented DNA (a feature of apoptotic cells) is end-labeled by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Immunohistochemical methods are then used to detect the labeled DNA and identify B-cell lymphoma cells in the last stage of apoptosis. Because the assay can lead to false-positive results, it is advisable to carry out an additional assay (e.g., immunohistochemistry for active caspase-3) to confirm the presence of apoptotic cells.

  10. Apoptosis-mediated seasonal testicular regression in the Japanese Jungle Crow (Corvus macrorhynchos).

    Science.gov (United States)

    Islam, M Nazrul; Tsukahara, N; Sugita, S

    2012-06-01

    The present study investigated effects of apoptosis observed during seasonal testicular regression in Japanese Jungle Crows. The study was conducted during January to June 2008, 2009. Testes from adults captured during non-breeding (January), prebreeding (February to mid-March), main-breeding (late March to early May), transition (mid-May to late May), and post-breeding (June) seasons were analyzed. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Paired-testis volume increased 95-fold from the non-breeding to the main-breeding season (P Crows; however, testis function was terminated rapidly after the breeding season. Furthermore, we concluded, similar to other avian species, Sertoli cell apoptosis followed by massive germ cell death was responsible for rapid testicular regression in Jungle Crows. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Growth inhibition and apoptosis induction by alternol in pancreatic carcinoma cells.

    Science.gov (United States)

    Cong, Pei-Fang; Qu, Ying-Chun; Chen, Jie-Peng; Duan, Li-Li; Lin, Cheng-Jiang; Zhu, Xiao-Lin; Li-Ling, Jesse; Zhang, Mei-Xia

    2015-04-21

    To investigate the effect of alternol on pancreatic cancer cells. Pancreatic cancer cells PANC-1 and BxPC3 were treated with various concentrations of alternol for 24, 48 and 72 h. Cell proliferation was measured by cell counting. Cell cycle distribution and mitochondrial membrane potential were determined by flow cytometry. Apoptosis was determined by a TdT-mediated dUTP nick end labeling assay and Hoechst staining. Expression of caspase 3, Bcl-2, p53 and p21 was measured by western blotting. Alternol showed dose- and time-dependent inhibition of the proliferation of PANC-1 and BxPC3 cells in vitro. Alternol induced apoptosis and cell cycle arrest at S phase and decreased mitochondrial membrane potential. Alternol activated caspase 3, upregulated p53 and p21 expression, and downregulated Bcl-2 expression in a dose-dependent manner. Our results suggested that alternol is a candidate for treatment of pancreatic cancer.

  12. A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index.

    Science.gov (United States)

    Hamidi, Jamal; Frainais, Christophe; Amar, Edouard; Bailly, Eric; Clément, Patrice; Ménézo, Yves

    2015-08-01

    The impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB.

  13. Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time.

    Science.gov (United States)

    Rougier, Natalia; Uriondo, Heydy; Papier, Sergio; Checa, Miguel Angel; Sueldo, Carlos; Alvarez Sedó, Cristian

    2013-07-01

    To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods. Comparative prospective study. Center for reproductive medicine. Twenty-seven semen samples from infertile patients. None. Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL ≥ 20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  14. Effects of topical human amniotic fluid and human serum in a mouse model of keratoconjunctivitis sicca.

    Science.gov (United States)

    Quinto, Guilherme G; Camacho, Walter; Castro-Combs, Juan; Li, Li; Martins, Suy Anne R; Wittmann, Priscila; Campos, Mauro; Behrens, Ashley

    2012-04-01

    To compare the effects of topical human amniotic fluid (HAF), topical human serum (HS), and topical artificial tears in a mouse model of dry eye. Thirty C57BL/6 mice were divided into 3 treatment groups: HAF, HS, and preservative-free artificial tears. Dry eye was induced by an injection of botulinum toxin B (BTX-B) into the lacrimal gland. Tear production and ocular surface fluorescein staining were evaluated in each mouse at 6 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Apoptotic keratocytes were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling test assay. A significant decrease in tear production was observed 3 days after BTX-B injection in all groups. At week 1, the HAF and HS groups had improved tear production compared with the control group (P < 0.001 and P = 0.003, respectively). HAF had a significantly improved fluorescein staining score compared with the HS (P = 0.043) and control (P = 0.007) groups at week 2. Goblet cell density was significantly decreased in the control group compared with the HAF and HS groups (P < 0.001). No difference in the amount of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive keratocytes was observed among the groups. HAF was superior to HS and artificial tears for improving corneal staining within 2 weeks of therapy in this induced mouse model of keratoconjunctivitis sicca. Clinical studies are needed to ascertain the benefits of these therapies in patients with ocular surface disorders associated with dry eye.

  15. Safety evaluation of poly(lactic-co-glycolic acid/poly(lactic-acid microspheres through intravitreal injection in rabbits

    Directory of Open Access Journals (Sweden)

    Rong XF

    2014-06-01

    Full Text Available Xianfang Rong,1 Weien Yuan,2 Yi Lu,1 Xiaofen Mo11Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China; 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: Poly(lactic-co-glycolic acid (PLGA and/or poly(lactic-acid (PLA microspheres are important drug delivery systems. This study investigated eye biocompatibility and safety of PLGA/PLA microspheres through intravitreal injection in rabbits. Normal New Zealand rabbits were randomly selected and received intravitreal administration of different doses (low, medium, or high of PLGA/PLA microspheres and erythropoietin-loaded PLGA/PLA microspheres. The animals were clinically examined and sacrificed at 1, 2, 4, 8, and 12 weeks postadministration, and retinal tissues were prepared for analysis. Retinal reactions to the microspheres were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end staining and glial fibrillary acidic protein immunohistochemistry. Retinal structure changes were assessed by hematoxylin and eosin staining and transmission electron microscopy. Finally, retinal function influences were explored by the electroretinography test. Terminal deoxynucleotidyl transferase-mediated dUTP nick end staining revealed no apoptotic cells in the injected retinas; immunohistochemistry did not detect any increased glial fibrillary acidic protein expression. Hematoxylin and eosin staining and transmission electron microscopy revealed no micro- or ultrastructure changes in the retinas at different time points postintravitreal injection. The electroretinography test showed no significant influence of scotopic or photopic amplitudes. The results demonstrated that PLGA/PLA microspheres did not cause retinal histological changes or functional damage and were biocompatible and safe enough for intravitreal injection in rabbits for controlled

  16. Green tea polyphenol epigallocatechin gallate inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes.

    Science.gov (United States)

    Lin, Ji; Della-Fera, Mary Anne; Baile, Clifton A

    2005-06-01

    Green tea catechins have been shown to promote loss of body fat and to inhibit growth of many cancer cell types by inducing apoptosis. The objective of this study was to determine whether epigallocatechin gallate (EGCG), the primary green tea catechin, could act directly on adipocytes to inhibit adipogenesis and induce apoptosis. Mouse 3T3-L1 preadipocytes and mature adipocytes were used. To test the effect of EGCG on viability, cells were incubated for 3, 6, 12, or 24 hours with 0, 50, 100, or 200 microM EGCG. Viability was quantitated by MTS assay. To determine the effect of EGCG on apoptosis, adipocytes were incubated for 24 hours with 0 to 200 microM EGCG, then stained with annexin V and propidium iodide and analyzed by laser scanning cytometry. Both preadipocytes and adipocytes were also analyzed for apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. To determine the effect of EGCG on adipogenesis, maturing preadipocytes were incubated during the 6-day induction period with 0 to 200 microM EGCG, then stained with Oil-Red-O and analyzed for lipid content. EGCG had no effect on either viability or apoptosis of preconfluent preadipocytes. EGCG also did not affect viability of mature adipocytes; however, EGCG increased apoptosis in mature adipocytes, as demonstrated by both laser scanning cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays. Furthermore, EGCG dose-dependently inhibited lipid accumulation in maturing preadipocytes. These results demonstrate that EGCG can act directly to inhibit differentiation of preadipocytes and to induce apoptosis of mature adipocytes and, thus, could be an important adjunct in the treatment of obesity.

  17. Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

    Science.gov (United States)

    Ubilla, A; Valdebenito, I; Árias, M E; Risopatrón, J

    2016-05-01

    In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Lider fallen zu wie die Tore der Burg : [luuletused kogust "Simunapäev"] / Kristiina Ehin ; aus dem Estnischen von Gisbert Jänicke

    Index Scriptorium Estoniae

    Ehin, Kristiina, 1977-

    2004-01-01

    Sisu: die nachricht von der sanktgeorgsnacht = Jüriöö sõnum ; sie standen unter einem schiefen dach = nad seisid viltuse katuse all ; "der soldat steht auf dem hof ..." = "püssimees seisab õues..." ; "der mond heult..." = "kuu ulub..." ; der frauenräuber = naisteröövija ; "ich schlafe als schlamm im wald..." = "magan mudana metsas..." ; "ruhig erhebt sich der weihrauchdunst..." = "vaikselt tõuseb taevasse..."

  19. The carving of the wood: an exhibition of work by David Pye, Nick Barberton, Matthew Burt, Gaynor Dowling, Eleanor Lakelin, Malcolm Martin and Keith Rand

    OpenAIRE

    Pulley, Bob; Glasgow, Andrew; Olding, Simon; Ratuszniak, Annette

    2014-01-01

    This catalogue was published and distributed by the Crafts Study Centre for the 2014 exhibition, The Carving of the Wood. The exhibition was curated and the accompanying catalogue was edited by Professor Simon Olding. The Crafts Study Centre holds important collections of wooden bowls and platters by David Pye, the celebrated Professor of Furniture Design at the Royal College of Art 1964-74. These works are presented at the heart of an exhibition which looks at the way that a group of ma...

  20. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    NARCIS (Netherlands)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase III alpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensa

  1. Lider fallen zu wie die Tore der Burg : [luuletused kogust "Simunapäev"] / Kristiina Ehin ; aus dem Estnischen von Gisbert Jänicke

    Index Scriptorium Estoniae

    Ehin, Kristiina, 1977-

    2004-01-01

    Sisu: die nachricht von der sanktgeorgsnacht = Jüriöö sõnum ; sie standen unter einem schiefen dach = nad seisid viltuse katuse all ; "der soldat steht auf dem hof ..." = "püssimees seisab õues..." ; "der mond heult..." = "kuu ulub..." ; der frauenräuber = naisteröövija ; "ich schlafe als schlamm im wald..." = "magan mudana metsas..." ; "ruhig erhebt sich der weihrauchdunst..." = "vaikselt tõuseb taevasse..."

  2. Nachtvögel. Nachtgedanken : Gedichte aus den Jahren 1995-1997 / Jaan Kaplinski ; tõlk. Gisbert Jänicke

    Index Scriptorium Estoniae

    Kaplinski, Jaan, 1941-

    1999-01-01

    Luuletused J. Kaplinski kogust "Öölinnud. Öömõtted". Tekst saksa ja eesti k. Sisu: "Über alles ist schon geschrieben, über alles schon gesungen..." = "Kõigest on kirjutatud, kõigest on lauldud..." ; "Die Blumen, durch die ich gesprochen..." = "Need lilled, mille läbi ma olen rääkinud..." ; "Du sagst, das Leichte besiegt am Ende immer das Schwere..." = "Sa ütlesid, et kergus võidab lõpuks ikka raskuse..." ; "In allem, was du jemals..." = "Kõiges, mis sa kunagi oled kirjutanud..." ; "Mondschöpfungsnacht. Ich wasche meine füsse im Dunkeln auf dem Steg. Warm ist es..." = "Kuuloomise öö. Pesen jalgu pimedas purdel. Soe..." ; "Immer mehr Autos, immer weniger Kühe..." = "Autosid ikka rohkem, lehmi vähem..."

  3. Die Form der Zeit : [aus "Aja kuju"¡ ("Die Form der Zeit")] : [luuletused] / Doris Kareva ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Kareva, Doris, 1958-

    2006-01-01

    Sisu: Die Form der Zeit : "Die lodernde, reglose Sonne..." ; "Was ist, kann ausgedrückt werden..." ; "Ich höre Stunde um Stunde..." ; "Ich ging am Strand entlang..." ; "Um sichtbarer zu leben..." Der beste Teil des Tags ist die Nacht..." ; "Nächte verschreiben sich dem Morgen..." ; "Herb, karg ist das Licht des Nordens..." ; "Alles rotiert, alles pulsiert..." ; "Sprache ist in der Tat das Haus des Seins..." ; "Der Schmetterling, dessen Flügelschlag..." ; "Die blendende, peinlich reine, bodenlose...". Orig.: Aja kuju : "Leekiv liikumatu päike..." ; "See, mis on, on väljendatav..." ; "Kuulan tunde ja tunde..." ; "Kõndisin mere ääres..." ; "Selleks, et elada selgemalt..." ; "Päeva parim osa on öö..." ; "Ööd kirjutavad end hommikuks..." ; "Karm, napp on põhjamaa valgus..." ; "Kõik tiirleb, kõik tuksleb..." ; "Keel on tõesti olemise koda..." ; "Liblikas, kelle tiivalöök..." ; "Lume pimestav, piinavalt puhas..."

  4. Synthesis, electronic structure, and magnetism of [Ni(6-Mes)2]+: a two-coordinate nickel(I) complex stabilized by bulky N-heterocyclic carbenes.

    Science.gov (United States)

    Poulten, Rebecca C; Page, Michael J; Algarra, Andrés G; Le Roy, Jennifer J; López, Isidoro; Carter, Emma; Llobet, Antoni; Macgregor, Stuart A; Mahon, Mary F; Murphy, Damien M; Murugesu, Muralee; Whittlesey, Michael K

    2013-09-18

    The two-coordinate cationic Ni(I) bis-N-heterocyclic carbene complex [Ni(6-Mes)2]Br (1) [6-Mes =1,3-bis(2,4,6-trimethylphenyl)-3,4,5,6-tetrahydropyrimidin-2-ylidene] has been structurally characterized and displays a highly linear geometry with a C-Ni-C angle of 179.27(13)°. Density functional theory calculations revealed that the five occupied metal-based orbitals are split in an approximate 2:1:2 pattern. Significant magnetic anisotropy results from this orbital degeneracy, leading to single-ion magnet (SIM) behavior.

  5. Production potential and stability of a broadleaved mixed oak/hornbeam forest stand situated on a eutrophic site, Ždánický les

    Directory of Open Access Journals (Sweden)

    Václav Hurt

    2012-01-01

    Full Text Available The paper focuses on assessing the growth and production of a mixed oak/hornbeam forest stand established by combined regeneration in 1940 to 1942. The stand is situated at an altitude of 460 m. Since 1961, it is left to its natural development. The 25–year–old stand was characterized as an individually mixed, both diameter- and height-differentiated pole-stage stand. The proportion of tree species was as follows: sessile oak 77 %, hornbeam 19 %, birch 1 %, lime 1 %, black poplar 1 %, wild cherry tree, wild service tree, and field maple. During 41 years of measurements, the proportion of oak slightly decreased to 76 %, on the other hand, the proportion of hornbeam increased to 22%. The initial growing stock of the 25–year–old stand, 75 m3.ha−1, increased to 323 m3.ha−1 at an age of 66 years in 2008. At present, current volume increment ranged between 6.3 m3.ha−1.year−1 and 11.6 m3.ha−1.year−1 during years 1967 and 1998. Since the age of 61, the growth of the stand has decreased and then even ceased due to increased mortality of oak.

  6. Die Form der Zeit : [aus "Aja kuju"¡ ("Die Form der Zeit")] : [luuletused] / Doris Kareva ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Kareva, Doris, 1958-

    2006-01-01

    Sisu: Die Form der Zeit : "Die lodernde, reglose Sonne..." ; "Was ist, kann ausgedrückt werden..." ; "Ich höre Stunde um Stunde..." ; "Ich ging am Strand entlang..." ; "Um sichtbarer zu leben..." Der beste Teil des Tags ist die Nacht..." ; "Nächte verschreiben sich dem Morgen..." ; "Herb, karg ist das Licht des Nordens..." ; "Alles rotiert, alles pulsiert..." ; "Sprache ist in der Tat das Haus des Seins..." ; "Der Schmetterling, dessen Flügelschlag..." ; "Die blendende, peinlich reine, bodenlose...". Orig.: Aja kuju : "Leekiv liikumatu päike..." ; "See, mis on, on väljendatav..." ; "Kuulan tunde ja tunde..." ; "Kõndisin mere ääres..." ; "Selleks, et elada selgemalt..." ; "Päeva parim osa on öö..." ; "Ööd kirjutavad end hommikuks..." ; "Karm, napp on põhjamaa valgus..." ; "Kõik tiirleb, kõik tuksleb..." ; "Keel on tõesti olemise koda..." ; "Liblikas, kelle tiivalöök..." ; "Lume pimestav, piinavalt puhas..."

  7. Der Handgänger : [aus "Kaitseala" ("Schutzgebiet")] : [luuletused] / Kristiina Ehin ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Ehin, Kristiina, 1977-

    2006-01-01

    Luuletusi tsüklist "Kätelkäija", kogumikust Kaitseala : Mohni 2004-2005 (Tallinn : Huma, 2005). Sisu: Der Handgänger ; "draussen ist üppiger juli..." ; "ich menschliche hülle keuche über die erde..." ; "die einsamen waldseen..." ; "wieso finden wir zueinander..." ; "die erste rätsel betrifft die wahrnehmung..." ; "die uhr schlägt für dich ind für mich die stunde des abschieds..." ; weisen, die mein herz dahintrugen 1.-2. Orig.: Kätelkäija ; "väljas on lopsakas juuli..." ; "ihulise inimesena hingeldan mööda maad..." ; "üksildased metsajärved..." ; "miks jõuame teineteiseni..." ; "esimene mõistatus puudutab taju..." ; "kell lööb minu ja sinu siit lahkumise tundi..." ; viisid mis viisid mu südame 1.-2.

  8. 青海某镍多金属矿选矿试验研究%Mineral processing of nicke polymetallic ore of Qinghai

    Institute of Scientific and Technical Information of China (English)

    胡志凯

    2015-01-01

    Studied at the nickel polymetallic ore of Qinghai,it contains 0.80% nickel,0.2% copper and 0.025%cobalt,that is a typical primary sulfide ore.The main copper mineral is chalcopyrite,pentlandite is the dominant nickel mineral,cobalt exist in pentlandite as isomorphism.The main gangue mineral is tremolite,talc and pyroxene.Process of tacle-copper nickel iso-flotation and nickel flotation was adopted. According to the closed circuit tests,copper concentration contains 25.15% copper,1.23% nickel at a 62.14% copper recover,meantime,nickel concentration contains 0.4% copper,0.24% cobalt and,7.95%nickel at a 80.67% nickel recovery and 75.76%cobalt recovery.%本文针对青海省某铜镍钴多金属矿进行选矿实验研究,该矿为原生硫化矿,原矿中的镍、铜、钴的含量分别为0.80%、0.20%、0.025%,铜矿物主要为黄铜矿,镍矿物主要为镍黄铁矿,钴以类质同象形式赋存在镍黄铁矿中,非金属矿物主要为透闪石、滑石、辉石。采用以铜为主的滑石-铜镍等可浮-尾矿强化回收镍工艺流程,闭路试验指标,铜精矿含铜25.15%,含镍1.23%,铜回收率62.14%;镍精矿含镍7.95%,含铜0.40%,含钴0.24%,镍回收率80.67%,钴回收率75.76%。

  9. [Ni(cod)2][Al(ORF)4], a Source for Naked Nickel(I) Chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Schwab, Miriam M. [Univ. of Freiburg (Germany). Institut fuer Anorganische und Analytische Chemie, Freiburger Materialforschungszentrum (FMF); Himmel, Daniel [Univ. of Freiburg (Germany). Institut fuer Anorganische und Analytische Chemie, Freiburger Materialforschungszentrum (FMF); Kacprzak, Sylwia [Univ. of Freiburg (Germany). Institut fuer Physikalische Chemie; Kratzert, Daniel [Univ. of Freiburg (Germany). Institut fuer Anorganische und Analytische Chemie, Freiburger Materialforschungszentrum (FMF); Radtke, Valentin [Univ. of Freiburg (Germany). Institut fuer Anorganische und Analytische Chemie, Freiburger Materialforschungszentrum (FMF); Weis, Philippe [Univ. of Freiburg (Germany). Institut fuer Anorganische und Analytische Chemie, Freiburger Materialforschungszentrum (FMF); Ray, Kallol [Humboldt Univ. of Berlin (Germany). Inst. fuer Chemie; Scheidt, Ernst-Wilhelm [Univ. of Ausburg (Germany). Inst. fuer Physik; Scherer, Wolfgang [Univ. of Ausburg (Germany). Inst. fuer Physik; de Bruin, Bas [Univ. of Amsterdam (Netherlands). Van‘t Hoff Inst. for Molecular Sciences (HIMS); Weber, Stefan [Univ. of Freiburg (Germany). Institut fuer Physikalische Chemie; Krossing, Ingo [Univ. of Freiburg (Germany). Institut fuer Anorganische und Analytische Chemie, Freiburger Materialforschungszentrum (FMF)

    2015-10-13

    The straightforward synthesis of the cationic, purely organometallic NiI salt [Ni(cod)2]+[Al(ORF)4]- was realized through a reaction between [Ni(cod)2] and Ag[Al(ORF)4] (cod=1,5-cyclooctadiene). Crystal-structure analysis and EPR, XANES, and cyclic voltammetry studies confirmed the presence of a homoleptic NiI olefin complex. Weak interactions between the metal center, the ligands, and the anion provide a good starting material for further cationic NiI complexes.

  10. Nachtvögel. Nachtgedanken : Gedichte aus den Jahren 1995-1997 / Jaan Kaplinski ; tõlk. Gisbert Jänicke

    Index Scriptorium Estoniae

    Kaplinski, Jaan, 1941-

    1999-01-01

    Luuletused J. Kaplinski kogust "Öölinnud. Öömõtted". Tekst saksa ja eesti k. Sisu: "Über alles ist schon geschrieben, über alles schon gesungen..." = "Kõigest on kirjutatud, kõigest on lauldud..." ; "Die Blumen, durch die ich gesprochen..." = "Need lilled, mille läbi ma olen rääkinud..." ; "Du sagst, das Leichte besiegt am Ende immer das Schwere..." = "Sa ütlesid, et kergus võidab lõpuks ikka raskuse..." ; "In allem, was du jemals..." = "Kõiges, mis sa kunagi oled kirjutanud..." ; "Mondschöpfungsnacht. Ich wasche meine füsse im Dunkeln auf dem Steg. Warm ist es..." = "Kuuloomise öö. Pesen jalgu pimedas purdel. Soe..." ; "Immer mehr Autos, immer weniger Kühe..." = "Autosid ikka rohkem, lehmi vähem..."

  11. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    NARCIS (Netherlands)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase III alpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is

  12. Inter- and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation.

    Science.gov (United States)

    Ribeiro, S; Sharma, R; Gupta, S; Cakar, Z; De Geyter, C; Agarwal, A

    2017-05-01

    One of the challenges with the sperm DNA fragmentation results is the inconsistency and the large variability in the results obtained by different techniques. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay quantifies the incorporation of fluoresceinated dUTP into single- and double-strand DNA breaks by labeling the 3'-OH terminal with TdT. The goal of this study was optimize the TUNEL protocol for assessment of sperm DNA fragmentation by standardization of the method and comparison of the data across two reference laboratories (i) at Basel, Switzerland and (ii) Cleveland Clinic, Ohio, USA. Semen samples from 31 subjects grouped into three cohorts. Sperm DNA fragmentation was data measured by two experienced operators at two different laboratories using identical semen samples, assay kit, protocol and acquisition settings using identical flow cytometers (BD Accuri C6). No significant differences were observed between the duplicates in any of the experiments performed. By including an additional washing step after fixation in paraformaldehyde, a high correlation was seen between the two laboratories (r = 0.94). A strong positive correlation was observed between the average sperm DNA fragmentation rates (r = 0.719). The mean sperm DNA fragmentation measured in each laboratory was similar. Both flow cytometers were identical in their settings and performance. This inter- and intra-laboratory study establishes that TUNEL is a reproducible assay when utilizing a standardized staining protocol and flow cytometer acquisition settings. Standardization and consensual guidelines for TUNEL validate the assay and establishes TUNEL as a robust test for measuring sperm DNA fragmentation especially in a multicenter setting. © 2017 American Society of Andrology and European Academy of Andrology.

  13. Leishmanicidal activities of Artemisia annua leaf essential oil against Visceral Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Mohammad eIslamuddin

    2014-11-01

    Full Text Available Visceral leishmaniasis (VL, the second-most dreaded parasitic disease after malaria, is currently endemic in 88 countries. Dramatic increases in the rates of infection, drug resistance and non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents from natural sources. In this study, we showed the leishmanicidal effect of essential oil from Artemisia annua leaves (AALEO against Leishmania donovani in vitro and in vivo. AALEO was extracted by hydrodistillation and characterized by GC-MS, the most abundant compounds were found to be camphor (52.06 % followed by β-caryophyllene (10.95 %. AALEO exhibited significant leishmanicidal activity against L. donovani, with 50 % inhibitory concentration of 14.63 ± 1.49 µg ml-1 and 7.3 ± 1.85 µg ml─1, respectively, against the promastigotes and intracellular amastigotes. The effect was mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labelling (TUNEL assay, dyskinetoplastidy, cell cycle arrest at sub-G0–G1 phase, loss of mitochondrial membrane potential and reactive oxygen species (ROS generation in promastigotes and nitric oxide (NO generation in ex vivo model. AALEO presented no cytotoxic effects against mammalian macrophages even at 200 µg ml─1. Intra-peritoneal administration of AALEO (200 mg/ kg.b.w. to infected BALB/c mice reduced the parasite burden by almost 90 % in the liver and spleen with significant reduction in weight. There was no hepato- or nephro-toxicity as demonstrated by normal levels of serum enzymes. The promising antileishmanial activity shown by camphor-rich AALEO may provide a new lead in the treatment of VL.

  14. Homocysteine Aggravates Cortical Neural Cell Injury through Neuronal Autophagy Overactivation following Rat Cerebral Ischemia-Reperfusion

    Directory of Open Access Journals (Sweden)

    Yaqian Zhao

    2016-07-01

    Full Text Available Elevated homocysteine (Hcy levels have been reported to be involved in neurotoxicity after ischemic stroke. However, the underlying mechanisms remain incompletely understood to date. In the current study, we hypothesized that neuronal autophagy activation may be involved in the toxic effect of Hcy on cortical neurons following cerebral ischemia. Brain cell injury was determined by hematoxylin-eosin (HE staining and TdT-mediated dUTP Nick-End Labeling (TUNEL staining. The level and localization of autophagy were detected by transmission electron microscopy, western blot and immunofluorescence double labeling. The oxidative DNA damage was revealed by immunofluorescence of 8-Hydroxy-2′-deoxyguanosine (8-OHdG. Hcy treatment aggravated neuronal cell death, significantly increased the formation of autophagosomes and the expression of LC3B and Beclin-1 in the brain cortex after middle cerebral artery occlusion-reperfusion (MCAO. Immunofluorescence analysis of LC3B and Beclin-1 distribution indicated that their expression occurred mainly in neurons (NeuN-positive and hardly in astrocytes (GFAP-positive. 8-OHdG expression was also increased in the ischemic cortex of Hcy-treated animals. Conversely, LC3B and Beclin-1 overexpression and autophagosome accumulation caused by Hcy were partially blocked by the autophagy inhibitor 3-methyladenine (3-MA. Hcy administration enhanced neuronal autophagy, which contributes to cell death following cerebral ischemia. The oxidative damage-mediated autophagy may be a molecular mechanism underlying neuronal cell toxicity of elevated Hcy level.

  15. N-Acetylcysteine counteracts oxidative stress and protects alveolar epithelial cells from lung contusion-induced apoptosis in rats with blunt chest trauma.

    Science.gov (United States)

    Topcu-Tarladacalisir, Yeter; Tarladacalisir, Taner; Sapmaz-Metin, Melike; Karamustafaoglu, Altemur; Uz, Yesim Hulya; Akpolat, Meryem; Cerkezkayabekir, Aysegul; Turan, Fatma Nesrin

    2014-08-01

    The aim of this study was to investigate the protective effects of N-acetylcysteine (NAC) on peroxidative and apoptotic changes in the contused lungs of rats following blunt chest trauma. The rats were randomly divided into three groups: control, contusion, and contusion + NAC. All the rats, apart from those in the control group, performed moderate lung contusion. A daily intramuscular NAC injection (150 mg/kg) was given immediately following the blunt chest trauma and was continued for two additional days following cessation of the trauma. Samples of lung tissue were taken in order to evaluate the tissue malondialdehyde (MDA) level, histopathology, and epithelial cell apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and active caspase-3 immunostaining. In addition, we immunohistochemically evaluated the expression of surfactant protein D (SP-D) in the lung tissue. The blunt chest trauma-induced lung contusion resulted in severe histopathological injury, as well as an increase in the MDA level and in the number of cells identified on TUNEL assay together with active caspase-3 positive epithelial cells, but a decrease in the number of SP-D positive alveolar type 2 (AT-2) cells. NAC treatment effectively attenuated histopathologic, peroxidative, and apoptotic changes, as well as reducing alterations in SP-D expression in the lung tissue. These findings indicate that the beneficial effects of NAC administrated following blunt chest trauma is related to the regulation of oxidative stress and apoptosis.

  16. Fermented nondigestible fraction from common bean (Phaseolus vulgaris L.) cultivar Negro 8025 modulates HT-29 cell behavior.

    Science.gov (United States)

    Cruz-Bravo, R K; Guevara-Gonzalez, R; Ramos-Gomez, M; Garcia-Gasca, T; Campos-Vega, R; Oomah, B D; Loarca-Piña, G

    2011-03-01

    The aim of the study was to evaluate the effect of a fermented nondigestible fraction (FNDF) of cooked bean (Phaseolus vulgaris L.) cultivar Negro 8025 on human colon adenocarcinoma HT-29 cell survival. Negro 8025 was chosen for in vitro fermentation based on comparison of chemical composition with 2 other cultivars: Azufrado Higuera and Pinto Durango. Negro 8025 had 58% total dietary fiber, 27% resistant starch, and 20 mg of (+)-catechin equivalents per gram of sample. Short-chain fatty acids (SCFAs) production and pH of the medium were measured after fermentation as indicators of colon protection through induced arrest on cell culture and apoptosis. Butyrate and pH of FNDF of Negro 8025 were higher than the control fermented raffinose extract. The FNDF inhibited HT-29 cell survival in a time- and concentration-dependent manner. The lethal concentration 50 (LC(50)) was 13.63% FNDF (equivalent to 7.36, 0.33, and 3.31 mmol of acetic, propionic, and butyric acids, respectively). DNA fragmentation, an apoptosis indicator, was detected by the TdT-mediated dUTP nick end labeling method in cells treated with the LC(50)-FNDF and a synthetic mixture of SCFAs mimicking LC(50)-FNDF. Our results suggest that common bean is a reliable source of fermentable substrates in colon, producing compounds with potential chemoprotective effect on HT-29 colon adenocarcinoma cells, so it may present an effective alternative to mitigate colon cancer development.

  17. Effect of green light spectra on the reduction of retinal damage and stress in goldfish, Carassius auratus.

    Science.gov (United States)

    Song, Jin Ah; Kim, Na Na; Choi, Young Jae; Choi, Cheol Young

    2016-07-22

    We investigated the effect of light spectra on retinal damage and stress in goldfish using green (530 nm) and red (620 nm) light emitting diodes (LEDs) at three intensities each (0.5, 1.0, and 1.5 W/m(2)). We measured the change in the levels of plasma cortisol and H2O2 and expression and levels of caspase-3. The apoptotic response of green and red LED spectra was assessed using the terminal transferase dUTP nick end labeling (TUNEL) assay. Stress indicator (cortisol and H2O2) and apoptosis-related genes (caspase-3) decreased in green light, but increased in red light with higher light intensities over time. The TUNEL assay revealed that more apoptotic cells were detected in outer nuclear layers after exposure to red LED over time with the increase in light intensity, than the other spectra. These results indicate that green light efficiently reduces retinal damage and stress, whereas red light induces it. Therefore, red light-induced retina damage may induce apoptosis in goldfish retina. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Increased apoptosis and decreased density of medial smooth muscle cells in human abdominal aortic aneurysms

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian张健; Jan Schmidt; Eduard Ryschich; Hardy Schumacher; Jens R Allenberg

    2003-01-01

    Objective To determine the increase of apoptosis and the decrease of smooth muscle cells (SMCs) density in human abdominal aortic aneurysms (AAA). Methods In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) was employed to detect apoptosis of SMCs in patients with AAA (n=25) and normal abdominal aortae (n=10). Positive cells were identified by specific cell marker in combination with immunohistochemistry. Meanwhile SMC counting was performed by anti-α-actin immunohistostaining to compare the SMC density. Results TUNEL staining revealed that there was significantly increased apoptosis in AAAs (average 8.6%) compared with normal abdominal aortae (average 0.95%, P<0.01). Double staining showed that most of these cells were SMCs. Counting of α-actin positive SMCs revealed that medial SMC density of AAAs (37.5±7.6 SMCs /HPF) was reduced by 79.1% in comparison with that of normal abdominal aortae (179.2±16.1 SMCs /HPF, P<0.01). Conclusions Significantly increased SMCs of AAA bear apoptotic markers initiating cell death. Elevated apoptosis may result in a decreased density of SMCs in AAA, which may profoundly influence the development of AAA.

  19. Abnormal development of tapetum and microspores induced by chemical hybridization agent SQ-1 in wheat.

    Directory of Open Access Journals (Sweden)

    Shuping Wang

    Full Text Available Chemical hybridization agent (CHA-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD, which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.

  20. Quercetin enhances TRAIL-induced apoptosis in prostate cancer cells via increased protein stability of death receptor 5

    Science.gov (United States)

    Jung, Young-Hwa; Heo, Jeonghoon; Lee, Yong J.; Kwon, Taeg Kyu; Kim, Young-Ho

    2010-01-01

    Aims Quercetin has been shown to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of prostate cancer cells via mechanisms that include upregulation of death receptor (DR) 5, a protein reported to play an important role in sensitizing cancer cells to apoptosis. We aimed to determine the specific mechanisms underlying quercetin-induced DR5 expression. Main methods Human prostate cancer cells were exposed to quercetin and TRAIL. Trypan blue assays and terminal transferase dUTP nick-end labeling (TUNEL) assays evaluated changes in TRAIL resistance after quercetin treatment, and flow cytometry examined quercetin-induced death receptor expression in DU-145 cells. Western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and transiently transfection were utilized to confirm apoptotic patterns of prostate cancer cells. Key findings After stimulation with quercetin, DU-145 cells exhibited stronger sensitization to TRAIL. Quercetin treatment enhanced TRAIL-induced activation proteins in the caspase pathway, such as poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-9. Quercetin dose-dependently increased DR5 levels in prostate cancer cells, which was mediated by increased transcription and protein stability, but not mRNA stability. Ectopic expression of DR5 dose-dependently increased TRAIL-induced apoptosis. Significance Our results showed that the role of quercetin and TRAIL combination therapy may provide a novel strategy for treating prostate cancer by overcoming critical mechanisms of apoptosis resistance. PMID:20096292

  1. Fas-FasL expression and myocardial cell apoptosis in patients with viral myocarditis.

    Science.gov (United States)

    Huang, T F; Wu, X H; Wang, X; Lu, I J

    2016-06-20

    The aim of the current study was to investigate Fas and FasL expression and myocardial cell apoptosis in viral myocarditis patients. Human heart specimens were selected from patients who were autopsied between February 2012 and February 2015; of these, 25 patients were diagnosed with viral myocarditis. Another 15 cases with no diagnosis of myocarditis were selected for the control group. All tissue specimens were divided into two parts, one for reverse transcription-polymerase chain reaction analysis and the other for immunohistochemical and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. In situ detection of apoptosis was performed by the TUNEL method, which revealed that myocardial cells from the viral myocarditis group exhibited significant apoptosis, whereas no apoptotic cells were observed in the control group. The number of cells staining positive for Fas and FasL protein in the viral myocarditis group was significantly higher than that in the control group (P myocarditis group than in the control group (P myocarditis. Furthermore, cytotoxic T lymphocytes may mediate cardiac muscle cells apoptosis via Fas-FasL signaling, and thus participate in the pathogenesis of viral myocarditis.

  2. Localization of the kappa opioid receptor gene to human chromosome band 8q11. 2

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    Yasuda, Kazuki; Takeda, Jun; Bell, G.I.; Espinosa, R.; Le Beau, M.M. (Univ. of Chicago, IL (United States))

    1994-02-01

    Using the cloned mouse kappa opioid receptor cDNA clone as a probe, screened a human genomic library and isolated a clone containing part of the human kappa opioid receptor gene (OPRK1), designated [lambda]hSR4-1. To determine the chromosomal localization of OPRK1, [lambda]hSR4-1 DNA was labeled with biotin by nick-translation in the presence of bio-11-dUTP and hybridized to human metaphase cells prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes as described previously. Hybridization of the OPRK1-specific probe [lambda]hSR4-1 DNA to normal human metaphase chromosomes resulted in specific labeling only of chromosome 8. Specific labeling of 8q11 was observed on all 4 (6 cells), 3 (9 cells), 2 (9 cells), or 1 (1 cell) chromatid of the chromosome 8 homologs in 25 cells examined. Of 72 signals observed, 70 were located at 8q11. 1 signal was located at 7q11 and at 12p11. In most cells, the signal on 8q was located at 8q11.2. 7 refs., 1 fig.

  3. Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes.

    Science.gov (United States)

    Fernández-Presas, Ana María; Tato, Patricia; Becker, Ingeborg; Solano, Sandra; Copitin, Natalia; Kopitin, Natalia; Berzunza, Miriam; Willms, Kaethe; Hernández, Joselin; Molinari, José Luis

    2010-05-01

    The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated.

  4. Deferasirox protects against iron-induced hepatic injury in Mongolian gerbil.

    Science.gov (United States)

    Al-Rousan, Rabaa M; Rice, Kevin M; Katta, Anjaiah; Laurino, Joseph; Walker, Ernest M; Wu, Miaozong; Triest, William E; Blough, Eric R

    2011-06-01

    Iron overload is associated with an increased risk of liver complications including fibrosis, cirrhosis, and hepatocellular carcinoma. Deferasirox is a new oral chelator with high iron-binding potency and selectivity. Here we investigate the ability of deferasirox to remove excessive hepatic iron and prevent iron-induced hepatic injury. Adult male Mongolian gerbils were divided into 3 groups (n=5/group)-control, iron overload (100 mg iron-dextran/kg body weight/5 days; intraperitoneal for 10 weeks), and iron overload followed by deferasirox treatment (100 mg deferasirox/kg body weight/d; pulse oral for 1 or 3 months). Compared with the nontreated iron overload group, deferasirox reduced hepatic iron concentration by 44% after 3 months of treatment (Pdeferasirox treatment, and no evidence of lipid accumulation was observed. Immunoblotting demonstrated that iron overload caused approximately 2-fold increase in hepatic ferritin expression (Pdeferasirox treatment (PDeferasirox treatment also was associated with reduced hepatic protein oxidation, superoxide abundance, and cell death. The percentage of terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells in the deferasirox-treated livers was 41% lower than that of iron overloaded group (Pdeferasirox treatment. These findings suggest that deferasirox may confer protection against iron-induced hepatic toxicity.

  5. Inhibition of Hepatocyte Apoptosis: An Important Mechanism of Corn Peptides Attenuating Liver Injury Induced by Ethanol.

    Science.gov (United States)

    Ma, Zhili; Hou, Tao; Shi, Wen; Liu, Weiwei; He, Hui

    2015-09-11

    In this study, the effects of mixed corn peptides and synthetic pentapeptide (QLLPF) on hepatocyte apoptosis induced by ethanol were investigated in vivo. QLLPF, was previously characterized from corn protein hydrolysis, which had been shown to exert good facilitating alcohol metabolism activity. Mice were pre-treated with the mixed corn peptides and the pentapeptide for 1 week and then treated with ethanol. After treatment of three weeks, the biochemical indices and the key ethanol metabolizing enzymes, the serum TNF-α, liver TGF-β1 concentrations and the protein expressions related to apoptosis were determined. We found that the Bcl-2, Bax and cytochrome c expressions in the intrinsic pathway and the Fas, FasL and NF-κB expressions in the extrinsic pathway together with higher TNF-α and TGF-β1 concentrations were reversed compared with the model group by both the mixed corn peptides and the pentapeptide. The activation of caspase3 was also suppressed. Additionally, apoptosis was further confirmed with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the TUNEL assay demonstrated peptides suppressed hepatocyte apoptosis. Our results suggest that apoptosis induced by ethanol is alleviated in response to the treatment of corn peptides, potentially due to reversing the related protein expression.

  6. Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats.

    Science.gov (United States)

    Roth, Steven; Dreixler, John C; Mathew, Biji; Balyasnikova, Irina; Mann, Jacob R; Boddapoti, Venkat; Xue, Lai; Lesniak, Maciej S

    2016-06-01

    We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. Eyes injected with hypoxic BMSC-conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect.

  7. Vitamin E and selenium treatment of monocrotaline induced hepatotoxicity in rats.

    Science.gov (United States)

    Cuce, G; Canbaz, H T; Sozen, M E; Yerlikaya, F H; Kalkan, S

    2017-01-01

    Monocrotaline (MCT) is a hepatotoxic pyrrolizidine alkaloid that is derived from plants; exposure may occur by consumption of contaminated grains, herbal teas and medicines. MCT can cause liver damage. We investigated the antioxidant effects of selenium (Se) and vitamin E against the toxic effects of MCT. Female Wistar albino rats were divided into four groups: a control group, an MCT group, an MCT + Se group, and an MCT + vitamin E group. Liver tissues were harvested, fixed, processed to paraffin and sections were cut. Anti-von Willebrand factor (vWF) immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL), and hematoxylin and eosin staining were performed. Serum and liver tissue glutathione (GSH), catalase (CAT), and glutathione peroxidase (GPx) levels were measured. Histopathological and TUNEL data showed significantly increased liver damage in the MCT group compared to controls. Histopathological and TUNEL staining indicated significant improvements in the MCT + vitamin E and MCT + Se groups compared to the MCT group. MCT significantly reduced the serum GSH level and GPx activity, and liver GPx activity. Biochemical data indicated a significant improvement in serum GSH level in the MCT + vitamin E group compared to the MCT group. We suggest that vitamin E and Se afford limited protection against MCT hepatotoxicity.

  8. Epidermal cell death in frogs with chytridiomycosis

    Science.gov (United States)

    Roberts, Alexandra A.; Skerratt, Lee F.; Berger, Lee

    2017-01-01

    Background Amphibians are declining at an alarming rate, and one of the major causes of decline is the infectious disease chytridiomycosis. Parasitic fungal sporangia occur within epidermal cells causing epidermal disruption, but these changes have not been well characterised. Apoptosis (planned cell death) can be a damaging response to the host but may alternatively be a mechanism of pathogen removal for some intracellular infections. Methods In this study we experimentally infected two endangered amphibian species Pseudophryne corroboree and Litoria verreauxii alpina with the causal agent of chytridiomycosis. We quantified cell death in the epidermis through two assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL) and caspase 3/7. Results Cell death was positively associated with infection load and morbidity of clinically infected animals. In infected amphibians, TUNEL positive cells were concentrated in epidermal layers, correlating to the localisation of infection within the skin. Caspase activity was stable and low in early infection, where pathogen loads were light but increasing. In animals that recovered from infection, caspase activity gradually returned to normal as the infection cleared. Whereas, in amphibians that did not recover, caspase activity increased dramatically when infection loads peaked. Discussion Increased cell death may be a pathology of the fungal parasite, likely contributing to loss of skin homeostatic functions, but it is also possible that apoptosis suppression may be used initially by the pathogen to help establish infection. Further research should explore the specific mechanisms of cell death and more specifically apoptosis regulation during fungal infection. PMID:28168107

  9. 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.

  10. Protective effect of inhalation of hydrogen gas on radiation-induced dermatitis and skin injury in rats.

    Science.gov (United States)

    Watanabe, Sadahiro; Fujita, Masanori; Ishihara, Masayuki; Tachibana, Shoichi; Yamamoto, Yoritsuna; Kaji, Tatsumi; Kawauchi, Toshio; Kanatani, Yasuhiro

    2014-11-01

    The effect of inhalation of hydrogen-containing gas (1.3% hydrogen + 20.8% oxygen + 77.9% nitrogen) (HCG) on radiation-induced dermatitis and on the healing of healing-impaired skin wounds in rats was examined using a rat model of radiation-induced skin injury. An X-ray dose of 20 Gy was irradiated onto the lower part of the back through two holes in a lead shield. Irradiation was performed before or after inhalation of HCG for 2 h. Inhalation of HCG significantly reduced the severity of radiodermatitis and accelerated healing-impaired wound repair. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and 8-hydroxy-2(')-deoxyguanosine (8-OHdG) showed that the proportion of apoptotic keratinocytes and the level of staining in the X-irradiated skin of rats that pre-inhaled HCG were significantly lower than that of rats which did not pre-inhale HCG. Cutaneous full-thickness wounds were then created in the X-irradiated area to examine the time-course of wound healing. X-irradiation significantly increased the time required for wound healing, but the inhalation of HCG prior to the irradiation significantly decreased the delay in wound healing compared with the control and post-inhalation of HCG groups. Therefore, radiation-induced skin injury can potentially be alleviated by the pre-inhalation of HCG.

  11. The neuroprotective agent Rasagiline mesylate attenuates cardiac remodeling after experimental myocardial infarction.

    Science.gov (United States)

    Varela, Aimilia; Mavroidis, Manolis; Katsimpoulas, Michalis; Sfiroera, Irini; Kappa, Niki; Mesa, Angelica; Kostomitsopoulos, Nikolaos G; Cokkinos, Dennis V

    2017-08-01

    Rasagiline mesylate (N-propargyl-1 (R)-aminoindan) (RG) is a selective, potent irreversible inhibitor of monoamine oxidase-B with cardioprotective and anti-apoptotic properties. We investigated whether it could be cardioprotective in a rat model undergoing experimental myocardial infarction (MI) by permanent ligation of the left anterior descending coronary artery. RG was administered, intraperitoneally, for 28 days (2 mg/kg) starting 24 h after MI induction. Echocardiography analysis revealed a significant reduction in left ventricular end-systolic and diastolic dimensions and preserved fractional shortening in RG-treated compared with normal saline group at 28 days post-MI (31.6 ± 2.3 vs. 19.6 ± 1.8, P < 0.0001), respectively. Treatment with RG prevented tissue fibrosis as indicated by interstitial collagen estimation by immunofluorescence staining and hydroxyproline content and attenuated the number of apoptotic myocytes in the border zone (65%) as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Caspase 3 relative protein levels were significantly decreased in the non-infarcted myocardium. Markedly decreased malondialdehyde levels in the border zone indicate a reduction in tissue oxidative stress. Our study demonstrates a positive effect of RG in the post-MI period with a significant attenuation in cardiac remodelling. © 2017 The Authors ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.

  12. Islet-1 is required for ventral neuron survival in Xenopus

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    Shi, Yu; Zhao, Shuhua; Li, Jiejing [CAS-Max Planck Junior Scientist Group, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Mao, Bingyu, E-mail: mao@mail.kiz.ac.cn [CAS-Max Planck Junior Scientist Group, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)

    2009-10-23

    Islet-1 is a LIM domain transcription factor involved in several processes of embryonic development. Xenopus Islet-1 (Xisl-1) has been shown to be crucial for proper heart development. Here we show that Xisl-1 and Xisl-2 are differentially expressed in the nervous system in Xenopus embryos. Knock-down of Xisl-1 by specific morpholino leads to severe developmental defects, including eye and heart failure. Staining with the neuronal markers N-tubulin and Xisl-1 itself reveals that the motor neurons and a group of ventral interneurons are lost in the Xisl-1 morphants. Terminal dUTP nick-end labeling (TUNEL) analysis shows that Xisl-1 morpholino injection induces extensive apoptosis in the ventral neural plate, which can be largely inhibited by the apoptosis inhibitor M50054. We also find that over-expression of Xisl-1 is able to promote cell proliferation and induce Xstat3 expression in the injected side, suggesting a potential role for Xisl-1 in the regulation of cell proliferation in co-operation with the Jak-Stat pathway.

  13. In Vitro Cytotoxicity of GuttaFlow Bioseal, GuttaFlow 2, AH-Plus and MTA Fillapex.

    Science.gov (United States)

    Saygili, Gokhan; Saygili, Suna; Tuglu, Ibrahim; Davut Capar, Ismail

    2017-01-01

    The aim of the present in vitro study was to evaluate the cytotoxicity of different sealers including GuttaFlow Bioseal, GuttaFlow 2, AH-Plus and MTA Fillapex on L929 murine fibroblasts. Samples of GuttaFlow Bioseal, GuttaFlow 2, AH-Plus and MTA Fillapex were fabricated in Teflon disks of 5 mm diameter and 3 mm thickness. L929 fibroblasts were exposed to the extracts of these materials for 3, 24, 72 and 168 h at 37(°)C with 5% CO2. Cell viability was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The data were analysed by ANOVA. GuttaFlow Bioseal was nontoxic at all experimental time points (P>0.05), whereas MTA Fillapex and AH-Plus were toxic (PMTA Fillapex was more cytotoxic than AH-Plus. There were more apoptotic cells in the MTA Fillapex and AH-Plus groups than in the other groups at 3 h (PMTA Fillapex and AH-Plus. At all experimental time points, there was no significant difference in the cell viability between the GuttaFlow Bioseal group and the control group.

  14. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality.

    Science.gov (United States)

    Takeda, Kumiko; Uchiyama, Kyoko; Kinukawa, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was determined. Individual differences in semen were detected using the sperm TUNEL index in these bulls (P bulls with a conception rate lower than 10%) and Holstein (n = 34) bulls were analyzed. The average sperm TUNEL index and conception rate resulting from artificial insemination (AI) were 4.7% and 55.7% for Japanese Black, and 4.9% and 39.5% for Holstein, respectively. A weak negative correlation between sperm TUNEL index and conception rate was observed in Holstein bulls (P bulls with more than 10% sperm TUNEL index were studied, and these samples showed low sperm viability. However, semen resulting in a very low conception rate did not have a high sperm TUNEL index. Although it would be difficult to predict a low conception rate resulting from AI using the sperm TUNEL index alone, the index can be used as an additional parameter to provide a more comprehensive description of semen quality.

  15. Caspase-Mediated Apoptosis in Sensory Neurons of Cultured Dorsal Root Ganglia in Adult Mouse

    Directory of Open Access Journals (Sweden)

    Hamid Reza Momeni

    2013-01-01

    Full Text Available Objective: Sensory neurons in dorsal root ganglia (DRG undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG.Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342 and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM and immunohistochemistry for activated caspase-3 were used.Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours.Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.

  16. Light-emitting-diode induced retinal damage and its wavelength dependency in vivo.

    Science.gov (United States)

    Shang, Yu-Man; Wang, Gen-Shuh; Sliney, David H; Yang, Chang-Hao; Lee, Li-Ling

    2017-01-01

    To examine light-emitting-diode (LED)-induced retinal neuronal cell damage and its wavelength-driven pathogenic mechanisms. Sprague-Dawley rats were exposed to blue LEDs (460 nm), green LEDs (530 nm), and red LEDs (620 nm). Electroretinography (ERG), Hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunohistochemical (IHC) staining, Western blotting (WB) and the detection of superoxide anion (O2(-)·), hydrogen peroxide (H2O2), total iron, and ferric (Fe(3+)) levels were applied. ERG results showed the blue LED group induced more functional damage than that of green or red LED groups. H&E staining, TUNEL, IHC, and TEM revealed apoptosis and necrosis of photoreceptors and RPE, which indicated blue LED also induced more photochemical injury. Free radical production and iron-related molecular marker expressions demonstrated that oxidative stress and iron-overload were associated with retinal injury. WB assays correspondingly showed that defense gene expression was up-regulated after the LED light exposure with a wavelength dependency. The study results indicate that LED blue-light exposure poses a great risk of retinal injury in awake, task-oriented rod-dominant animals. The wavelength-dependent effect should be considered carefully when switching to LED lighting applications.

  17. Albumin resuscitation protects against traumatic/hemorrhagic shock-induced lung apoptosis in rats

    Institute of Scientific and Technical Information of China (English)

    Yun ZHANG; Zhong-yan LIANG; Shao-yang ZHANG; Fang-fang HUANG; Wei WU; Yuan GAO; Zuo-bing CHEN

    2008-01-01

    Objective: To determine the effects of albumin administration on lung injury and apoptosis in traumatic/hemorrhagic shock (T/HS) rats. Methods: Studies were performed on an in vivo model of spontaneously breathing rats with induced T/HS; the rats were subjected to femur fracture, ischemia for 30 min, and reperfusion for 20 rain with Ringer's lactate solution (RS) or 5% (w/v) albumin (ALB), and the left lower lobes of the lungs were resected. Results: Albumin administered during reperfusion markedly attenuated injury of the lung and decreased the concentration of lactic acid and the number of in situ TdT-mediated dUTP nick-end labelling (TUNEL)-positive cells. Moreover, immunohistochemistry performed 24 h after reperfusion revealed increases in the level of nuclear factor κB (NF-κB), and phosphorylated p38 mitogen-activated protein kinase (MAPK) in the albumin-untreated group was down-regulated by albumin treatment when compared with the sham rats. Conclusion: Resuscitation with albumin attenuates tissue injury and inhibits T/HS-induced apoptosis in the lung via the p38 MAPK signal transduction pathway that functions to stimulate the activation of NF-κB.

  18. Protective effect of daidzin against D-galactosamine and lipopolysaccharide-induced hepatic failure in mice.

    Science.gov (United States)

    Kim, Sung-Hwa; Heo, Jeong-Haing; Kim, Yeong Shik; Kang, Sam Sik; Choi, Jae Sue; Lee, Sun-Mee

    2009-05-01

    This study examined the effects of daidzin, a major isoflavone from Puerariae Radix, on D-galactosamine (D-GalN) and lipopolysaccharide (LPS)-induced liver failure. Mice were given an intraperitoneal injection of daidzin (25, 50, 100 and 200 mg/kg) 1 h before receiving an injection of D-GalN (700 mg/kg)/LPS (10 microg/kg). Daidzin markedly reduced the elevated serum aminotransferase activity and the levels of lipid peroxidation and tumor necrosis factor-alpha. The glutathione content was lower in the D-GalN/LPS group, which was attenuated by daidzin. The daidzin pretreatment attenuated the swollen mitochondria observed in the d-GalN/LPS group. Daidzin attenuated the apoptosis of hepatocytes, which was confirmed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and a caspase-3 assay. Overall, these results suggest that the liver protection of daidzin is due to reduced oxidative stress and its antiapoptotic activity.

  19. Study on Relationship between the Thickness of Tongue Fur and the Expressions of Apoptosis-related Genes of the Tongue Epithelial Cells in Patients with Diseases of the Digestive System

    Institute of Scientific and Technical Information of China (English)

    Wu Zhengzhi; Li Ming; Zhang Yongfeng; Chen Manyin

    2007-01-01

    To investigate the relationship between the thickness of tongue fur, apoptosis of the tongue fur epithelial cells and expressions of apoptosis-related genes in diseases of the digestive system,apoptosis-related genes TGF-β3, fas mRNA and protein products were detected with terminal deoxynucleotidyl transferase-mediated deoxyurine triphosphate (d-UTP) nick-end labeling(TUNEL)technique, in situ hybridization, immunohistochemical methods, and image analysis technique,respectively. Results indicated that compared with the normal tongue fur, over-expression of fas gene was found in the peeling fur with an increase in cell apoptosis, while a low-expression of TGF-β3 in the thick fur with a decrease in cell apoptosis. The changes in expression levels of fas and TGF-β3 genes,apoptosis-promoting genes in the tongue fur epithelial cells, had a similar tendency of cell apoptosis level.It is concluded that the changes in expression levels of fas and TGF-β3 are possibly important reasons influencing apoptosis of epithelial cells of tongue fur and leading to changes in thickness of the tongue fur.

  20. S-phase kinase-associated protein 2 knockdown blocks colorectal cancer growth via regulation of both p27 and p16 expression.

    Science.gov (United States)

    Xu, S-Y; Wang, F; Wei, G; Wang, B; Yang, J-Y; Huang, Y-Z; Zhang, L; Zheng, F; Guo, L-Y; Wang, J-N; Tang, J-M

    2013-12-01

    The objective of this study was to determine the role and mechanism of S-phase kinase-associated protein 2 (Skp2) in colorectal cancer cell proliferation and survival both in vitro and in vivo. Adenoviral vector expressing Skp2 short hairpin RNA was transduced into SW480 cells. The effects of Skp2 on cell cycle and survival were assessed by Flow Cytometry. Cell proliferation was analyzed by MTT assay. The expression of cell cycle regulators p16 and p27 were measured by western blot. In vivo, human colorectal cancer was produced by xenograft of cancer cells in nude mouse. Tumor growth inhibitory rate was calculated to generate growth curve. Tumor growth was monitored by examining proliferating cell nuclear antigen expression, whereas tumor cell apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL) staining. Knockdown of Skp2 blocked SW480 tumor cell growth and induced cell apoptosis. Skp2 appeared to be very important for the progression of cell cycle at G1/S phase. In vivo, blockade of Skp2 expression inhibited tumor growth and induced tumor apoptosis. Mechanistically, Skp2 regulated the expression of both p27 and p16 both in vitro and in vivo. The conclusion that we derive from this study is that Skp2 regulates colorectal cancer cell growth by inhibiting the expression of cell cycle regulator p27 and p16.

  1. Cadmium-induced ectopic apoptosis in zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Po Kwok; Cheng, Shuk Han [Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon (Hong Kong)

    2003-02-01

    In this study, we tested the hypothesis that cadmium-induced developmental toxicity was mediated via ectopic occurrence of apoptosis during embryonic development. We employed confocal microscopy to acquire images of whole-mount staining of apoptotic cells in zebrafish embryo exposed to 100 {mu}M cadmium from 5 hours post fertilisation (hpf) to 28 hpf. Three-dimensional reconstruction of the images was performed and the spatial and temporal distributions of apoptotic cells in the embryos were compared. In cadmium-treated embryos with varying degrees of gross developmental malformations, significantly higher numbers of apoptotic cells were detected with this method. In order to detect the precise locations of apoptotic cells, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay in sectioned embryos. In the degenerating neural tube of cadmium-treated embryos apoptotic cells were detected, while in the healthy neural tube of the untreated controls no apoptotic cells were found. We then employed flow cytometry to investigate whether cadmium exposure would affect the dynamics of apoptosis or induce any abnormalities in cell-cycle progression. It appeared that cadmium did not induce cell-cycle arrest. The percentages of apoptotic cells did not differ in the two groups at 13, 16 or 19 hpf. At 28 hpf, however, a significantly higher percentage of apoptotic cells were found in the cadmium-treated group. Exposure to cadmium, therefore, induced ectopic apoptosis at 28 hpf without affecting the dynamics of apoptosis at earlier developmental stages. (orig.)

  2. Fas/Fas ligand-mediated apoptosis in different cell lineages and functional compartments of human lymph nodes.

    Science.gov (United States)

    Kokkonen, Tuomo S; Karttunen, Tuomo J

    2010-02-01

    We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL-mediated apoptosis in lymph node homeostasis.

  3. Fas/Fas Ligand–mediated Apoptosis in Different Cell Lineages and Functional Compartments of Human Lymph Nodes

    Science.gov (United States)

    Kokkonen, Tuomo S.; Karttunen, Tuomo J.

    2010-01-01

    We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL–mediated apoptosis in lymph node homeostasis. (J Histochem Cytochem 58:131–140, 2010) PMID:19826071

  4. Neuropeptide Treatment with Cerebrolysin Enhances the Survival of Grafted Neural Stem Cell in an α-Synuclein Transgenic Model of Parkinson’s Disease

    Science.gov (United States)

    Rockenstein, Edward; Desplats, Paula; Ubhi, Kiren; Mante, Michael; Florio, Jazmin; Adame, Anthony; Winter, Stefan; Brandstaetter, Hemma; Meier, Dieter; Moessler, Herbert; Masliah, Eliezer

    2015-01-01

    Neuronal stem cell (NSC) grafts have been investigated as a potential neuro-restorative therapy in Parkinson’s disease (PD) but their use is compromised by the death of grafted cells. We investigated the use of Cerebrolysin (CBL), a neurotrophic peptide mixture, as an adjunct to NSC therapy in the α-synuclein (α-syn) transgenic (tg) model of PD. In vehicle-treated α-syn tg mice, there was decreased survival of NSCs. In contrast, CBL treatment enhanced the survival of NSCs in α-syn tg groups and ameliorated behavioral deficits. The grafted NSCs showed lower levels of terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells in the CBL-treated mice when compared with vehicle-treated α-syn tg mice. No evidence of tumor growth was detected. Levels of α-syn were similar in the vehicle in CBL-treated tg mice. In conclusion, CBL treatment might be a potential adjuvant for therapeutic NSC grafting in PD. PMID:27429559

  5. Protective effect of prostaglandin E₁ on radiation-induced proliferative inhibition and apoptosis in keratinocytes and healing of radiation-induced skin injury in rats.

    Science.gov (United States)

    Takikawa, Megumi; Sumi, Yuki; Tanaka, Yoshihiro; Nambu, Masaki; Doumoto, Takashi; Yanagibayashi, Satoshi; Azuma, Ryuichi; Yamamoto, Naoto; Kishimoto, Satoko; Ishihara, Masayuki; Kiyosawa, Tomoharu

    2012-01-01

    We examined the effects of prostaglandin E₁ (PGE₁) on radiation-induced proliferation inhibition and apoptosis in keratinocytes and healing of radiation-induced skin injury in a rat model. PGE₁ had a protective effect on radiation-induced growth inhibition in keratinocytes in vitro, but not in fibroblasts. Varying concentrations of PGE₁ were subcutaneously administered into the posterior neck region. X-irradiation at a dose of 20 Gy was administrated to the lower part of the back using a lead sheet with two holes 30 min to 1 h before or after the administration of PGE₁. Although X-irradiation induced epilation, minor erosions, or skin ulcers in almost all rats, PGE₁ administration prior to irradiation reduced these irradiation injuries. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling showed that proportions of apoptotic keratinocytes in the X-irradiated skin of PGE₁-administered rats were significantly lower than for those in the skin of rats which did not receive PGE₁. Cutaneous full-thickness defective wounds were then formed in X-irradiated areas to examine the time course of wound healing. Wound healing was significantly delayed because of X-irradiation, but PGE₁ administration prior to irradiation led to a significantly shorter delay in wound healing compared with controls. Decreasing delay in wound healing was correlated with concentration of PGE₁ administrated. Thus, PGE₁-administration may potentially alleviate the radiation-induced skin injury.

  6. Exogenous surfactant suppresses inflammation in experimental endotoxin-induced lung injury.

    Science.gov (United States)

    Mittal, Neha; Sanyal, Sankar Nath

    2009-01-01

    Our objective was to evaluate the anti-inflammatory effects of exogenous surfactant and surfactant phospholipids on the lipopolysaccharide (LPS)-induced lung injury. Exogenous surfactant (porcine surfactant) and surfactant phospholipid (dipalmitoyl phospholipid DPPC, hexadecanol, tylaxopol) were instilled intratracheally with LPS in rats. Expression of surfactant apoproteins (SP-A) and the cyclooxygenase enzymes (COX-1 and -2) was studied by immunohistochemistry, and apoptosis was analyzed by in situ terminal dUTP nick end labeling TUNEL assay. The intracellular reactive oxygen species (ROS) was measured in the isolated macrophages by fluorescence measurement with dichlorofluorescein diacetate (DCFH-DA). LPS-induced oxidative burst and apoptosis at 72 hours were reduced by both porcine and synthetic surfactant. SP-A as well as COX-1 and -2 expressions were suppressed with synthetic surfactant treatment, whereas with porcine surfactant (P-SF) the SP-A expression was enhanced in response to LPS administration. These results indicate that exogenous surfactant inhibits LPS-induced inflammation. This anti-inflammatory activity may be an important outcome of surfactant therapy in endotoxin-induced respiratory distress.

  7. The Root Extract of Gentiana macrophylla Pall. Alleviates Cardiac Apoptosis in Lupus Prone Mice.

    Directory of Open Access Journals (Sweden)

    Chih-Yang Huang

    Full Text Available The roots of the perennial herb Gentiana macrophylla Pall. (GM are known as Qinjiao, which has been used for centuries to treat systemic lupus erythematosus (SLE. However, little is known about the effects of GM on cholesterol-aggravated cardiac abnormalities in SLE, and the mechanisms thereof. This study investigates whether GM exhibits anti-apoptotic effects, focusing on the left ventricle (LV of NZB/W F1 mice fed with high-cholesterol diet. The morphology and apoptotic status of ventricular tissues were determined by microscopy and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. Levels of apoptotic biomarkers were determined by immunoblotting. The results thus obtained revealed that GM significantly reduced the cholesterol-aggravated apoptosis of LV in NZB/W F1 mice by suppressing both intrinsic and extrinsic apoptotic pathways. Additionally, GM significantly increased the cardiac insulin-like growth factors (IGF-1 survival signaling and anti-apoptotic proteins in LV tissues. Accordingly, GM is considered to be beneficial in alleviating cholesterol-aggravated cardiac damage in SLE, and therefore constitute an alternative treatment for SLE patients with cardiac abnormalities.

  8. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2016-01-01

    Full Text Available Genistein is effective against amyloid-β toxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer's disease. A rat model of Alzheimer's disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-β peptide (25–35. In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg was given prior to establishing Alzheimer's disease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genist-ein in Alzheimer's disease rats. These findings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

  9. Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining.

    Science.gov (United States)

    Daniel, Bron; DeCoster, Mark A

    2004-08-01

    The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain is in wide use for measuring apoptosis in neurons, as well as in other cell types. TUNEL may give false positive results due to variations in labeling technique as well as staining of cells that have undergone non-apoptotic DNA strand breaks. Therefore, in isolation, TUNEL is not a certain indicator of apoptosis. Recently, we have demonstrated the potent apoptotic effect of secreted phospholipase A2 from group III (sPLA2-III) on primary cortical neurons from rat. Here we describe a computer-assisted method for quantifying TUNEL-positive neurons after sPLA2-III induced apoptosis. Extent of TUNEL is normalized to total nuclear content using 4',6-diamidino-2-phenylindole (DAPI) staining. Furthermore, DAPI counterstaining allows for determination of a nuclear morphology indicator, based on nuclear size and roundness, which we call the nuclear area factor. We found that the nuclear area factor is an early indicator of cell death (significant after 4 h post treatment), while TUNEL staining is significant at later times (26 h). Thus, the independent staining techniques using TUNEL and DAPI complement each other, and with commercially available image analysis software, may be used to indicate early as well as delayed cell injury processes.

  10. Levistolide A overcomes P-glycoprotein-mediated drug resistance in human breast carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Fei CHEN; Tao WANG; Jia WANG; Zi-qiang WANG; Ming QIAN

    2008-01-01

    Aim:The aim of the present study was to investigate the reversing effect of levistolide A (LA) on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in human breast carcinoma Bcap37/MDR1 cells. Methods:After chemotherapeu-tic drugs (adriamycin or vincristine) used alone or in combination with LA, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-lium bromide assay and cell cycle distribution by flow cytometry. RT-PCR was used to detect MDR1 gene transcription and the Western blot assay was used to assess P-gp expression and the cleavages of poly(ADP-ribose) polymerase and caspase-3. Apoptosis was detected by terminal transferase-mediated dUTP nick end-labeling assay. Moreover, the P-gp function was evaluated by the intracellu-lar accumulation of the P-gp substrate detected by flow cytometry. Results:We found the subcytotoxic doses of LA significantly enhanced adriamycin- or vinc-ristine-induced G2/M arrest and apoptosis. These effects were consistent with the ability of LA to inhibit P-gp function. Moreover, LA dramatically enhanced the verapamil (VER) ability to reverse drug resistance. Conclusion:LA has the poten-tial to be developed as a novel P-gp modulator. Furthermore, the combination of LA and VER might represent a more sufficient but less toxic anti-MDR regimen.

  11. THE ROLES OF bcl-2 GENE FAMILY IN THE PULMONARY ARTERY REMODELING OF HYPOXIA PULMONARY HYPERTENSION IN RATS

    Institute of Scientific and Technical Information of China (English)

    杨成; 王胜发; 梁桃; 王巨; 王凯; 王柏春

    2001-01-01

    Objective. To investigate the roles of apoptosis in the pulmonary artery remodeling of pulmonary hypertension secondary to hypoxia and illustrate the relative genes expression.Methods. Thirty rats were divided into hypoxia group(10%O2, 8h/d) and normal control group. On the 15th day of hypoxia, pulmonary artery pressure and right ventricular hypertrophy index were measured and pulmonary artery vessels were studied by light microscope. Then terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)technique was used to detect nucleosomal DNA fragmentation of apoptotic cells.In situ hybridization and RT-PCR were used to detect the expression level of bcl-2 and bax.``Results. The pulmonary artery pressure and right ventricular hypertrophy index of hypoxia group were increased significantly, the pulmonary artery wall of hypoxic group become incrassate than control group. Apoptotic cells can be found in lung with hypoxia or without hypoxia. Compared with control group, apoptotic index of hypoxic group decreased significantly. Through the methods of in situ hybridization and RT-PCR, we found the expression of bcl-2 increased whereas bax decreased significantly in the hypoxic group.``Conclusion. The alternation in bcl-2 and bax expression induced by hypoxia play an important role in the pulmonary artery remodeling which is the main pathologic change of pulmonary hypertension secondary to hypoxia.

  12. Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Xia, D.Y. [Department of Neurology, Navy General Hospital of PLA, Beijing (China); Li, W. [General Hospital of Shenyang Military Command, Department of Neurology, Shenyang, China, Department of Neurology, General Hospital of Shenyang Military Command, Shenyang (China); Qian, H.R.; Yao, S.; Liu, J.G.; Qi, X.K. [Department of Neurology, Navy General Hospital of PLA, Beijing (China)

    2013-08-10

    Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.

  13. Action and mechanism of Fas and Fas ligand in immune escape of gallbladder carcinoma

    Science.gov (United States)

    Xu, Li-Ning; Zou, Sheng-Quan; Wang, Jian-Ming

    2005-01-01

    AIM: To study the role of Fas and Fas ligand (FasL) in biological behaviors of gallbladder carcinoma, and their correlated action and mechanism in tumor escape. METHODS: Streptavidin-biotin-peroxidase immunohisto-chemistry technique was used to study the expression of Fas and FasL protein in 26 gallbladder carcinoma tissues, 18 gallbladder adenoma tissues, 3 gallbladder dysplasia tissues and 20 chronic cholecystitis tissues. Apoptosis of the infiltrating lymphocytes in these tissues was studied by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Expression of both proteins and apoptosis of the tumor infiltrating lymphocytes in cancer tissues of primary foci was compared with clinicopathological features of gallbladder carcinoma. RESULTS: The positive rates of Fas were not significantly different among carcinoma, adenoma, dysplasia and chronic cholecystitis. The positive rate of FasL in carcinoma was significantly higher than that in chronic cholecystitis (χ2 = 4.89, P0.05). Apoptosis of infiltrating lymphocytes was not discovered in adenoma and chronic cholecystitis. CONCLUSION: FasL expressed in gallbladder carcinoma cells permits tumor cells to escape from immune surveillance of organism by inducing apoptosis in infiltrating lymphocytes of carcinoma tissues. Up-regulation of FasL expression plays an important role in invasive depth, histological classification and metastasis of gallbladder carcinoma. PMID:15968727

  14. Neuroprotective effects of lycopene pretreatment on transient global cerebral ischemia‑reperfusion in rats: The role of the Nrf2/HO‑1 signaling pathway.

    Science.gov (United States)

    Lei, Xiaofeng; Lei, Lijian; Zhang, Zhelin; Cheng, Yan

    2016-01-01

    The present study aimed to investigate the neuroprotective effect of lycopene in a mouse model of bilateral common carotid artery occlusion (BCCAO) and the role of the Nrf2/HO‑1 signaling pathway. A total of 60 male C57BL/6 mice, aged 12 weeks and weighing 20‑24 g, were used in the present study. The mice were randomly assigned to three groups: Control, BCCAO and BCCAO + lycopene. The neurological score was assessed 24, 48 or 72 h following BCCAO. Hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were performed to detect neuronal death and survival. The production of glutathione (GSH) and reactive oxygen species were detected to investigate the oxidative stress. The expression levels of nuclear factor erythroid 2‑related factor (Nrf2) and Heme oxygenase‑1 (HO‑1) were determined by western blotting. Lycopene significantly improved the neurological score in the BCCAO mice. It attenuated neuronal apoptosis, as indicated by TUNEL staining, and attenuated the oxidative stress induced by global ischemia. Lycopene increased the expression levels of Nrf2 and HO‑1, indicating that the Nrf2/HO‑1 signaling pathway may be involved in the neuroprotective effect of lycopene. The present study revealed that lycopene protects the brain from global ischemic injury, which is associated with its antiapoptotic effect and the activation of the Nrf2/HO‑1 signaling pathway.

  15. Lycopene induces apoptosis in Candida albicans through reactive oxygen species production and mitochondrial dysfunction.

    Science.gov (United States)

    Choi, Hyemin; Lee, Dong Gun

    2015-08-01

    Lycopene, a well-known carotenoid pigment found in tomatoes, has shown various biological functions. In our previous report, we showed that lycopene induces two apoptotic hallmarks, plasma membrane depolarization and G2/M cell cycle arrest, in Candida albicans. In this study, we investigated the ability of lycopene to induce apoptosis, and the mechanism by which it regulates apoptosis. FITC-Annexin V staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, and 4',6-diamidino-2-phenylindole (DAPI) assay showed that lycopene exerted its antifungal activity during the early and late stages of apoptosis in C. albicans. During apoptosis, intracellular reactive oxygen species (ROS) were increased, and specifically the hydroxyl radicals contributed to the fungal cell death. Furthermore, lycopene treatment caused intracellular Ca(2+) overload and mitochondrial dysfunction, such as mitochondrial depolarization and cytochrome c release from the mitochondria to the cytoplasm. At last caspase activation was triggered. In summary, lycopene exerted its antifungal effects against C. albicans by inducing apoptosis via ROS production and mitochondrial dysfunction.

  16. Apoptosis of pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid in combination with horseradish peroxidase

    Institute of Scientific and Technical Information of China (English)

    Chen Huang; Lu-Sheng Si; Li-Ying Liu; Tu-Sheng Song; Lei Ni; Ling Yang; Xiao-Yan Hu; Jing-Song Hu; Li-Ping Song; Yu Luo

    2005-01-01

    AIM: To explore the mechanisms underlying the apoptosis of human pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid (TAA) in combination with horseradish peroxidase (HRP).METHODS: BXPC-3 cells derived from human pancreatic cancer were exposed to 40 or 80 μmol/L IAA and 1.2 μg/mL HRP at different times. Then, MTT assay was used to detect the cell proliferation. Flow cytometry was performed to analyze cell cycle. Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling assay was used to detect apoptosis. 2,7-Dichlorofluorescin diacetate uptake was measured by confocal microscopy to determine free radicals. Level of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods.RESULTS: IAA/HRP initiated growth inhibition of BXPC-3 cells in a dose- and time-dependent manner. Flow cytometry revealed that the cells treated for 48 h were arrested at G1/G0. After exposure to 80 μmol/L IAA plus 1.2 μg/mL HRP for 72 h, the apoptosis rate increased to 72.5‰,which was nine times that of control. Content of MDA and activity of SOD increased respectively after treatment compared to control. Meanwhile, IAA/HRP stimulated the formation of free radicals.CONCLUSION: The combination of IAA and HRP can inhibit the growth of human pancreatic cancer BXPC-3 cells in vitro by inducing apoptosis.

  17. Detection of cell apoptosis with TUNEL method after infection of canine distemper virus%应用缺口末端标记法(TUNEL)分析犬瘟热病毒(CDV)诱导的细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    郭爱珍; 陆承平

    2000-01-01

    利用犬瘟热病毒(canine distemper virus,CDV)弱毒标准株OP-CDV感染敏感细胞系非洲绿猴肾细胞(Vero),应用缺口末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)分析感染细胞的凋亡.结果在感染后48 h的Vero细胞中检出了凋亡阳性细胞,而对照细胞内未检测到凋亡细胞.感染后24 h的细胞尚未出现细胞病变,这一时期的细胞内也未检测到凋亡细胞.表明CDV能诱导细胞调亡,且凋亡细胞的检出时间与细胞病变的出现时间呈正向相关.

  18. Rolipram stimulates angiogenesis and attenuates neuronal apoptosis through the cAMP/cAMP-responsive element binding protein pathway following ischemic stroke in rats.

    Science.gov (United States)

    Hu, Shouye; Cao, Qingwen; Xu, Peng; Ji, Wenchen; Wang, Gang; Zhang, Yuelin

    2016-03-01

    Rolipram, a phosphodiesterase-4 inhibitor, can activate the cyclic adenosine monophosphate (cAMP)/cAMP-responsive element binding protein (CREB) pathway to facilitate functional recovery following ischemic stroke. However, to date, the effects of rolipram on angiogenesis and cerebral ischemia-induced neuronal apoptosis are yet to be fully elucidated. In this study, the aim was to reveal the effect of rolipram on the angiogenesis and neuronal apoptosis following brain cerebral ischemia. Rat models of ischemic stroke were established following transient middle cerebral artery occlusion and rolipram was administered for three, seven and 14 days. The results were examined using behavioral tests, triphenyl tetrazolium chloride staining, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to evaluate the effects of rolipram therapy on functional outcome, angiogenesis and apoptosis. Western blot analysis was used to show the phosphorylated- (p-)CREB protein level in the ischemic hemisphere. The rolipram treatment group exhibited a marked reduction in infarct size and modified neurological severity score compared with the vehicle group, and rolipram treatment significantly promoted the microvessel density in the ischemic boundary region and increased p-CREB protein levels in the ischemic hemisphere. Furthermore, a significant reduction in the number of TUNEL-positive cells was observed in the rolipram group compared with the vehicle group. These findings suggest that rolipram has the ability to attenuate cerebral ischemic injury, stimulate angiogenesis and reduce neuronal apoptosis though the cAMP/CREB pathway.

  19. Anticancer Effect of Curcumin on B Cell non- Hodgkin's Lymphoma

    Institute of Scientific and Technical Information of China (English)

    SUN Chunyan; LIU Xinyue; CHEN Yan; LIU Fang

    2005-01-01

    To explore the anticancer effect of curcumin on human B cell non-Hodgkin's lymphoma and compare its effects on human B cell non-Hodgkin's lymphoma cells and normal peripheral blood mononuclear cells (NPBMNCs). MTT assay was used to study the effect of curcumin on the growth of Raji cells and NPBMNCs. The effect of curcumin on the apoptosis of Raji cells and NPBMNC were studied by flow cytometry and TDT-mediated dUTP nick and labeling (TUNEL). The effect of curcumin on the cell cycle of Raji cells were examined by propidium iodide staining flow cytometry. The results showed that curcumin strongly inhibited ±1.82 μmol/L and curcumin induced Raji cell apoptosis in a time- and dose-dependent manner. Raji cells treated with curcumin showed curcumin did not demonstrate apparent proliferation inhibition and apoptosis induction in NPBMNCs. It was concluded that curcumin is able to inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Morever, curcumin has low toxicity on NPBMNCs but can selectively induce apoptosis in Raji cells.

  20. Effect of cholecystokinin on learning and memory, neuronal proliferation and apoptosis in the rat hippocampus

    Science.gov (United States)

    Reisi, Parham; Ghaedamini, Ali Reza; Golbidi, Mohammad; Shabrang, Moloud; Arabpoor, Zohreh; Rashidi, Bahman

    2015-01-01

    Background: Cholecystokinin (CCK) has roles in learning and memory, but the cellular mechanism is poorly understood. This study investigated the effect of CCK on spatial learning and memory, neuronal proliferation and apoptosis in the hippocampus in rats. Materials and Methods: Experimental groups were control and CCK. The rats received CKK octapeptide sulfated (CCK-8S, 1.6 μg/kg, i.p.) for 14 days. Spatial learning and memory were tested by Morris water maze and finally immunohistochemical study was performed; neurogenesis by Ki-67 method and apoptosis by Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) assay in hippocampal dentate gyrus (DG). Results: Cholecystokinin increased Ki-67 positive cells and reduced TUNEL positive cells in the granular layer of hippocampal DG. CCK failed to have a significant effect on spatial learning and memory. Conclusion: Results indicate neuroprotective and proliferative effects of CCK in the hippocampus; however, other factors are probably involved until the newly born neurons achieve necessary integrity for behavioral changes. PMID:26623402

  1. Gold namoprtices enhance anti-tumor effect of radiotherapy to hypoxic tumor

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Mi Sun; Lee, Eun Jung; Kim, Jae Won; Keum, Ki Chang; Koom, Woong Sub [Dept. of Radiation Oncology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Chung, Ui Seok; Koh, Won Gun [Dept. of Chemical and Biomolecular Engineering, Yonsei University, Seoul (Korea, Republic of)

    2016-09-15

    Hypoxia can impair the therapeutic efficacy of radiotherapy (RT). Therefore, a new strategy is necessary for enhancing the response to RT. In this study, we investigated whether the combination of nanoparticles and RT is effective in eliminating the radioresistance of hypoxic tumors. Gold nanoparticles (GNPs) consisting of a silica core with a gold shell were used. CT26 colon cancer mouse model was developed to study whether the combination of RT and GNPs reduced hypoxia-induced radioresistance. Hypoxia inducible factor-1α (HIF-1α) was used as a hypoxia marker. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were conducted to evaluate cell death. Hypoxic tumor cells had an impaired response to RT. GNPs combined with RT enhanced anti-tumor effect in hypoxic tumor compared with RT alone. The combination of GNPs and RT decreased tumor cell viability compare to RT alone in vitro. Under hypoxia, tumors treated with GNPs + RT showed a higher response than that shown by tumors treated with RT alone. When a reactive oxygen species (ROS) scavenger was added, the enhanced antitumor effect of GNPs + RT was diminished. In the present study, hypoxic tumors treated with GNPs + RT showed favorable responses, which might be attributable to the ROS production induced by GNPs + RT. Taken together, GNPs combined with RT seems to be potential modality for enhancing the response to RT in hypoxic tumors.

  2. Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the “ladder pattern” revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner.The number of TUNEL-positive cells was dramatically increased to 33.6±1.2% from 2.8±0.3% by treat ment with heparin in different concentrations (10~40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2,bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations.These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

  3. Differential routes of carboplatin administration influence lymphocyte apoptosis in retroperitoneal lymph nodes.

    Science.gov (United States)

    Huang, Yong-Wen; Zeng, Zheng; Li, Su; Liu, Ji-Hong

    2012-12-01

    We aimed to investigate carboplatin distribution in retroperitoneal lymph nodes and its effect on lymphocyte apoptosis following intravenous (IV), intra-arterial (IA), and retroperitoneal (RP) administration. Sixty-three healthy female canines were randomly assigned as IV, IA, or RP administration of carboplatin. At 0.5, 1, 2, 4, 8, 24, and 72 h after carboplatin treatment, retroperitoneal lymph nodes (n = 6 at each time point) were collected and high-performance liquid chromatography was employed to measure the carboplatin content. The differences in carboplatin pharmacokinetics of the three administration routes were compared. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was carried out to measure the lymphocyte apoptosis of the retroperitoneal lymphocytes. The peak concentration of carboplatin in plasma following IV administration was the highest among all approaches; as to the peak time, RP administration was longer than the other two administrations. Concentration for carboplatin in the retroperitoneal lymph node was highest following IA administration at early time points, but at higher time points, concentration was significantly higher following RP administration. Penetration of carboplatin into the retroperitoneal space was higher following RP administration. Following RP administration, the level of apoptotic lymphocytes in the retroperitoneal lymph nodes was significantly greater than either IV or IA. Following RP administration of carboplatin, the concentration, area under the curve of carboplatin and the number of apoptotic lymphocytes were significantly higher than those following IV and IA administration. This suggests that RP administration of carboplatin is beneficial for the treatment of retroperitoneal lymph node metastasis.

  4. Retinal protective effects of topically administered agmatine on ischemic ocular injury caused by transient occlusion of the ophthalmic artery

    Directory of Open Access Journals (Sweden)

    S. Hong

    2012-03-01

    Full Text Available Agmatine, an endogenous polyamine and putative neuromodulator, is known to have neuroprotective effects on various neurons in the central nervous system. We determined whether or not topically administered agmatine could reduce ischemic retinal injury. Transient ocular ischemia was achieved by intraluminal occlusion of the middle cerebral artery of ddY mice (30-35 g for 2 h, which is known to also induce occlusion of the ophthalmic artery. In the agmatine group (N = 6, a 1.0 mM agmatine-containing ophthalmic solution was administered four times daily for 2 weeks before occlusion. In the control group (N = 6, a 0.1% hyaluronic acid ophthalmic solution was instilled at the same times. At 22 h after reperfusion, the eyeballs were enucleated and the retinal sections were stained by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL. Transient ocular ischemia induced apoptosis of retinal cells in the entire retinal layer, and topically administered agmatine can significantly reduce this ischemic retinal injury. The proportion of apoptotic cells was definitely decreased (P < 0.001; Kruskal-Wallis test. Overall, we determined that topical agmatine application effectively decreases retinal damage in an in vivo ocular ischemic injury model. This implies that agmatine is a good candidate as a direct neuroprotective agent for eyes with ocular ischemic diseases.

  5. Ulinastatin suppresses endoplasmic reticulum stress and apoptosis in the hippocampus of rats with acute paraquat poisoning

    Directory of Open Access Journals (Sweden)

    Hai-feng Li

    2015-01-01

    Full Text Available Lung injury is the main manifestation of paraquat poisoning. Few studies have addressed brain damage after paraquat poisoning. Ulinastatin is a protease inhibitor that can effectively stabilize lysosomal membranes, prevent cell damage, and reduce the production of free radicals. This study assumed that ulinastatin would exert these effects on brain tissues that had been poisoned with paraquat. Rat models of paraquat poisoning were intraperitoneally injected with ulinastatin. Simultaneously, rats in the control group were administered normal saline. Hematoxylin-eosin staining showed that most hippocampal cells were contracted and nucleoli had disappeared in the paraquat group. Fewer cells in the hippocampus were concentrated and nucleoli had disappeared in the ulinastatin group. Western blot assay showed that expressions of GRP78 and cleaved-caspase-3 were significantly lower in the ulinastatin group than in the paraquat group. Immunohistochemical findings showed that CHOP immunoreactivity was significantly lower in the ulinastatin group than in the paraquat group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining showed that the number of apoptotic cells was reduced in the paraquat and ulinastatin groups. These data confirmed that endoplasmic reticular stress can be induced by acute paraquat poisoning. Ulinastatin can effectively inhibit this stress as well as cell apoptosis, thereby exerting a neuroprotective effect.

  6. Cutaneous eccrine glands of the foot pads of the rock hyrax (Procavia capensis, Hyracoidea, Mammalia).

    Science.gov (United States)

    Stumpf, P; Welsch, U

    2002-01-01

    In order to find correlations between skin gland morphology and specific ethological features, the cutaneous glands of the foot pads of Procavia capensis were studied by histological and various histochemical methods and by electron microscopy. In the foot pads, abundant specific eccrine skin glands occur, which consist of coiled tubular secretory portions and coiled ducts. The wall of the secretory part is composed of cuboidal glandular cells and myoepithelial cells. Among the glandular cells two types occur: clear and dark cells. Clear cells have numerous mitochondria and form a basal labyrinth, indicating fluid transport. Dark cells, which stain strongly with periodic acid-Schiff, contain a highly developed perinuclear Golgi apparatus, large amounts of rough endoplasmic reticulum and many secretory granules indicating production of glycoproteins. Cytokeratin (CK) 19 was found in secretory compartments and ducts, CK14 only in duct cells. Single cells of the secretory coils and ducts may be stained with antibodies against antimicrobial peptides. Some glandular cells contain proliferating cell nuclear antigen-positive nuclei especially in the ducts indicating an increased cell proliferation. Terminal transferase (TdT)-mediated d-UTP nick-end labeling-positive nuclei can be detected predominantly in the secretory coils and rarely in the transitional portions between ducts and end pieces. We suppose that proliferating cells migrate from the ducts to the secretory coils. The secretory product of the eccrine cutaneous glands seems to improve the traction between the foot pads of these animals and the steep and smooth rock formations among which they live.

  7. Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model

    Directory of Open Access Journals (Sweden)

    Azza A. Al-Mahrouki

    2014-03-01

    Full Text Available Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67, blood leakage (factor VIII, angiogenesis (cluster of differentiation molecule CD31, ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF], oxygen limitation (prolyl hydroxylase PHD2 and DNA damage/repair (γH2AX. Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy.

  8. Aqueous extract of Cordyceps alleviates cerebral ischemia-induced short-term memory impairment in gerbils.

    Science.gov (United States)

    Lee, Sang-Hak; Ko, Il-Gyu; Kim, Sung-Eun; Hwang, Lakkyong; Jin, Jun-Jang; Choi, Hyun-Hee; Kim, Chang-Ju

    2016-04-01

    Cerebral ischemia is caused by reduced cerebral blood flow due to a transient or permanent cerebral artery occlusion. Ischemic injury in the brain leads to neuronal cell death, and eventually causes neurological impairments. Cordyceps, the name given to the fungi on insects, has abundant useful natural products with various biological activities. Cordyceps is known to have nephroprotective, hepatoprotective, anti-inflammatory, antioxidative, and antiapoptotic effects. We investigated the effects of Cordyceps on short-term memory, neuronal apoptosis, and cell proliferation in the hippocampal dentate gyrus following transient global ischemia in gerbils. For this study, a step-down avoidance test, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, immunohistochemistry for caspase-3 and 5-bromo-2'-de-oxyuridine, and western blot for Bax, Bcl-2, brain-derived neurotrophic factor (BDNF), and tyrosin kinase B were performed. In the present study, Cordyceps alleviated cerebral ischemia-induced short-term memory impairment. Cordyceps showed therapeutic effects through inhibiting cerebral ischemia-induced apoptosis in the hippocampus. Cordyceps suppressed cerebral ischemia-induced cell proliferation in the hippocampal dentate gyrus due to the reduced apoptotic neuronal cell death. Cordyceps treatment also enhanced BDNF and TrkB expressions in the hippocampus of ischemic gerbils. It can be suggested that Cordyceps overcomes cerebral ischemia-induced neuronal apoptosis, thus facilitates recovery following cerebral ischemia injury.

  9. Inhibitory effects of oleoylethanolamide (OEA) on H₂O₂-induced human umbilical vein endothelial cell (HUVEC) injury and apolipoprotein E knockout (ApoE-/-) atherosclerotic mice.

    Science.gov (United States)

    Ma, Li; Guo, Xiaobing; Chen, Wei

    2015-01-01

    Atherosclerosis (AS) is initiated by vascular endothelial cell injury, which is induced by lipid and protein oxidation. Oleoylethanolamide (OEA), a dietary fat-derived lipid, has shown atheroprotective effect. In vitro studies demonstrated that OEA showed cytoprotective effects on H2O2-induced primary cultured human umbilical vein endothelial cell (HUVEC) injury model. Further investigation of the cytoprotective effects of OEA demonstrated that OEA exerted its function by scavenging for reactive oxygen species, as well as increasing anti-oxidative enzymes, reducing lipid peroxidation, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and apoptosis-related proteins expression. The in vivo study using an ApoE-/- mouse model fed with high-fat diet for 8 weeks showed that OEA (10 mg/kg/day, i.g.) administration reduced blood lipid levels, prevented endothelial cell damage and inhibited early AS plaque formation. In conclusion, our results suggested that OEA exerted a pharmacological effect on ameliorating atherosclerotic plaque formation through the inhibition of oxidative stress-induced endothelial cell injury and therefore OEA can be a potential candidate drug for anti-atherosclerosis.

  10. Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines by Echinophora platyloba DC: In Vitro Analysis

    Directory of Open Access Journals (Sweden)

    Fatemeh Zare Shahneh

    2013-01-01

    Full Text Available Echinophora platyloba DC plant (Khousharizeh is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared from Echinophora platyloba DC, on mouse fibrosarcoma cell line (WEHI-164. Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT- mediated dUTP nick end labeling (TUNEL assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 μg/mL when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So the Echinophora platyloba DC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway.

  11. Programmed cell death during terminal bud senescence in a sympodial branching tree,Eucommia ulmoides

    Institute of Scientific and Technical Information of China (English)

    XU Wenjie; Kalima-N'Koma MWANGE; CUI Keming

    2004-01-01

    Eucommia ulmoides Oliv. is a typical sympodial branching tree. The apical bud of the branch ages and dies every year, replaced by the nearby axillary bud in the second year. Structural assays and a series of biochemical analyses were performed to analyze the senescence mechanism in the apical bud. It was revealed that most cells of the apical bud underwent the programmed cell death (PCD) during the senescence: the chromosomes were congregated and the nuclear contents were condensed, as shown by 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. DNA fragmentation was detected during senescence using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end in situ labeling (TUNEL) method, coincident with the appearance of a DNA ladder. Moreover, a 20 kD DNase related to fragmentation was found. PCD was initiated first in the young leaves, leaf primordia and peripheral zone cells, then in the central mother cells and initial layer cells in the apical meristem. The terminal buds remain in vegetative growth during senescence, in contrast to buds of many annual plants.

  12. The Ameliorative Effects of L-2-Oxothiazolidine-4-Carboxylate on Acetaminophen-Induced Hepatotoxicity in Mice

    Directory of Open Access Journals (Sweden)

    Jun Ho Shin

    2013-03-01

    Full Text Available The aim of the study was to investigate the ameliorative effects and the mechanism of action of L-2-oxothiazolidine-4-carboxylate (OTC on acetaminophen (APAP-induced hepatotoxicity in mice. Mice were randomly divided into six groups: normal control group, APAP only treated group, APAP + 25 mg/kg OTC, APAP + 50 mg/kg OTC, APAP + 100 mg/kg OTC, and APAP + 100 mg/kg N-acetylcysteine (NAC as a reference control group. OTC treatment significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels in a dose dependent manner. OTC treatment was markedly increased glutathione (GSH production and glutathione peroxidase (GSH-px activity in a dose dependent manner. The contents of malondialdehyde and 4-hydroxynonenal in liver tissues were significantly decreased by administration of OTC and the inhibitory effect of OTC was similar to that of NAC. Moreover, OTC treatment on APAP-induced hepatotoxicity significantly reduced the formation of nitrotyrosin and terminal deoxynucleotidyl transferase dUTP nick end labeling positive areas of liver tissues in a dose dependent manner. Furthermore, the activity of caspase-3 in liver tissues was reduced by administration of OTC in a dose dependent manner. The ameliorative effects of OTC on APAP-induced liver damage in mice was similar to that of NAC. These results suggest that OTC has ameliorative effects on APAP-induced hepatotoxicity in mice through anti-oxidative stress and anti-apoptotic processes.

  13. Mechanism of Mitochondrial Connexin43′s Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Shuai Hou

    2016-05-01

    Full Text Available We observed mitochondrial connexin43 (mtCx43 expression under cerebral ischemia-reperfusion (I/R injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO. Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD activity and malondialdehyde (MDA content. MtCx43, p-mtCx43, protein kinase C (PKC, and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX and diazoxide (DZX groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists.

  14. Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs).

    Science.gov (United States)

    Takikawa, Megumi; Ishihara, Masayuki; Takabayashi, Yuki; Sumi, Yuki; Takikawa, Makoto; Yoshida, Ryuichi; Nakamura, Shingo; Hattori, Hidemi; Yanagibayashi, Satoshi; Yamamoto, Naoto; Kiyosawa, Tomoharu

    2015-04-13

    The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.

  15. Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injur y

    Institute of Scientific and Technical Information of China (English)

    Guo-zhu Sun; Fen-fei Gao; Zong-mao Zhao; Hai Sun; Wei Xu; Li-wei Wu; Yong-chang He

    2016-01-01

    Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endo-plasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of lfuid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediat-ed dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These ifndings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.

  16. Knockdown of Stat3 expression using RNAi inhibits growth of laryngeal tumors in vivo

    Institute of Scientific and Technical Information of China (English)

    Li-fang GAO; Lian-ji WEN; Hao YU; Ling ZHANG; Yan MENG; Yue-ting SHAO; De-qi XU; Xue-jian ZHAO

    2006-01-01

    Aim:To study the effect of pSilencer1.0-U6-siRNA-stat3 on the growth of human laryngeal tumors in nude mice.Methods:Hep2 cells were transplanted into nude mice,then at the time of tumor fornaation,growth rates were observed.After the tumor formed,pSilencer1.0-U6-siRNA-stat3 was injected.Tumor volumes were calculated,and growth curves were plotted.Representative histological sections were taken from mice beating transplantation tumors in both treated and control groups,and stat3,Ptyr-star3,Bcl-2,cyclin D1,and survivin expression were detected by Western blotting.survivin Mrna levels were detected by Northern blotting,hematoxylin and cosin staining and terminal deoxvribonucleotidvl transferase-mediated Dutp-digoxigenin nick end-1abeling (TUNEL)assay to confirm the apoptosis of tumors.Results:In nude mice,pSilencer1.0-U6-siRNA-stat3 significantly suppressed the growth of tumors compared with controls (P<0.001). It suppressed stat3 expression,and downregulated BcL2,cyclin D1,and survivin expression within the tumor.This significantly induced apoptosis of the tumors.Conclusion:pSilencer1.0-U6-siRNA-stat3 was able to inhibit the growth of transplanted human laryngeal tumors in nude mice and induce apoptosis.

  17. Neuropeptide Treatment with Cerebrolysin Enhances the Survival of Grafted Neural Stem Cell in an α-Synuclein Transgenic Model of Parkinson's Disease.

    Science.gov (United States)

    Rockenstein, Edward; Desplats, Paula; Ubhi, Kiren; Mante, Michael; Florio, Jazmin; Adame, Anthony; Winter, Stefan; Brandstaetter, Hemma; Meier, Dieter; Moessler, Herbert; Masliah, Eliezer

    2015-01-01

    Neuronal stem cell (NSC) grafts have been investigated as a potential neuro-restorative therapy in Parkinson's disease (PD) but their use is compromised by the death of grafted cells. We investigated the use of Cerebrolysin (CBL), a neurotrophic peptide mixture, as an adjunct to NSC therapy in the α-synuclein (α-syn) transgenic (tg) model of PD. In vehicle-treated α-syn tg mice, there was decreased survival of NSCs. In contrast, CBL treatment enhanced the survival of NSCs in α-syn tg groups and ameliorated behavioral deficits. The grafted NSCs showed lower levels of terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells in the CBL-treated mice when compared with vehicle-treated α-syn tg mice. No evidence of tumor growth was detected. Levels of α-syn were similar in the vehicle in CBL-treated tg mice. In conclusion, CBL treatment might be a potential adjuvant for therapeutic NSC grafting in PD.

  18. Prevention of diabetic microangiopathy by prophylactic transplant of mobilized peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Bin ZHOU; Xiao-cang CAO; Zhi-hong FANG; Cui-lin ZHENG; Zhi-bo HAN; He REN; Man-chiu POON; Zhong-chao HAN

    2007-01-01

    Aim: To investigate whether the prophylactic local delivery of mobilized periph-eral blood mononuclear cells (M-PBMNC) could prevent peripheral microangio-pathy in diabetic nude mice. Methods: Diabetic nude mice were induced with intraperitoneal injections of streptozotocin. With the time course of diabetes, we detected the capillary and arteriole density of mice adductor muscles by immuno-histopathy. In situ apoptosis was detected by using TdT-mediated dUTP nick end labeling (TUNEL) methods. M-PBMNC were labeled and locally delivered to the adductor muscles. Mononuclear cells were also isolated and cultured in vitro for the detection and counting of endothelial progenitor cells(EPC). Results: Rarefication of capillaries and arterioles, enhanced apoptosis in adductor muscles,and reduced circulating EPC in diabetic nude mice. Prophylactic local delivery of M-PBMNC halted the progression of microvascular rarefaction in hind-limb skel-etal muscles by inhibiting apoptosis. We detected the survival, migration and incorporation of transplanted M-PBMNC into the murine vasculature in vivo. In addition, more EPC were available from M-PBMNC than non-mobilized cells.Conclusion: These results suggested that the prophylactic local delivery of M-PBMNC may represent a novel approach for the treatment of microvascular complications in diabetics.

  19. Matrine Activates PTEN to Induce Growth Inhibition and Apoptosis in V600EBRAF Harboring Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Shuiying Wang

    2013-07-01

    Full Text Available Here, we report a natural chemical Matrine, which exhibits anti-melanoma potential with its PTEN activation mechanism. Matrine effectively inhibited proliferation of several carcinoma cell lines, including melanoma V600EBRAF harboring M21 cells. Flow cytometry analysis showed Matrine induced G0/G1 cell cycle arrest in M21 cells dose-dependently. Apoptosis in M21 cells induced by Matrine was identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL analysis and Annexin-V/FITC staining. Molecular mechanistic study suggested that Matrine upregulated both mRNA level and protein expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN, leading to inhibition of the PI3K/Akt pathway. Downregulation of phosphor-Aktser473 by Matrine activated p21 and Bax, which contributed to G0/G1 cell cycle and apoptosis. Besides, Matrine enhanced the PI3K/Akt inhibition effects to inhibit the cell proliferation with PI3K inhibitor, LY2940002. In summary, our findings suggest Matrine is a promising antitumor drug candidate with its possible PTEN activation mechanisms for treating cancer diseases, such as melanomas.

  20. Bilobalide inhibits 6-OHDA-induced activation of NF-κB and loss of dopaminergic neurons in rat substantia nigra

    Institute of Scientific and Technical Information of China (English)

    Ling-yun LI; Xi-lin ZHAO; Xi-feng FEI; Zhen-lun GU; Zheng-hong QIN; Zhong-qin LIANG

    2008-01-01

    Aim: To investigate the offect of bilobalide on the activation of NF-κB, and apoptasis of dopaminergic neurons induced by 6-hydroxydopamine (6-OHDA). Methods: A rat model of Parkinson's disease was produced with a unilateral infu-sion of 6-OHDA (8 μg) into the substantia nigra par compact. Bilobalide was administered 5, 10, and 20 mg/kg (ip) once a day for 7 d, starting 6 d prior to the 6-OHDA infusion. The rats were subjected to locomotor activity and rotational behavior testing 2 or 3 weeks after the 6-OHDA infusion. The expressions of tyrosine hydroxylase (TH) and NF-κB p65 were examined by immunofluorescence. The loss of dopaminergic neurons was detected by Nissl's staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to iden-tify apoptosis. ResUltS; Tho behavioral changes due to 6-OHDA were signifi-cantly restored by bilobalide pretreatment. Bilobalide inhibited the 6-OHDA-induced loss of TH-positive neurons, decreased the activation of NF-κB, and protected dopaminergic neurons from apoptosis remarkably. Conclusion: NF-κB activation contributes to the 6-OHDA-induced loss of dopaminergic neurons, and the inhibition of the NF-κB pathway is likely to be involved in the neuroprotective effect of bilobalide.

  1. Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

    Institute of Scientific and Technical Information of China (English)

    Jialong GUO; Kailun ZHANG; Yanmei JI; Xionggang JIANG; Shunqing ZUO

    2008-01-01

    In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups (n=8 in each group): control group was perfused for 120min. In the I/R group, after 30min stabilization the injury was induced by 30min global ischemia followed by 60min reperfusion. Ethyl pyruvate (EP) group was set up with the same protocol as I/R group except that it was supplied with 2mmol/L EP 15min before ischemia and throughout reperfusion. Myocardial malonaldehyde (MDA) content Was measured. Myocardial apoptotic index (AI) was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group (P<0.05). These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

  2. Antioxidative and Anti-Apoptotic Roles of Silibinin in Reversing Learning and Memory Deficits in APP/PS1 Mice.

    Science.gov (United States)

    Bai, Dafeng; Jin, Ge; Yin, Shiliang; Zou, Dan; Zhu, Qiwen; Yang, Zhihang; Liu, Xuan; Ren, Lizheng; Sun, Yifeng; Gan, Shiming

    2017-08-29

    Silibinin has been widely used to treat liver diseases due to its antioxidant activity. However, the effects of silibinin on the central nervous system have not been thoroughly investigated. The pathological hallmarks of Alzheimer's disease are the accumulation of amyloid β protein, development of neurofibrillary tangles and increased oxidative stress, which ultimately lead to irreversible neuronal loss and cognitive impairment. Our findings show that silibinin ameliorated memory impairments in APP/PS1 mice in the Morris water maze via suppression of oxidative stress and inhibition of apoptosis. Treatment with silibinin reduced malondialdehyde content level and increased glutathione and superoxide dismutase activity in APP/PS1 mice. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay revealed an anti-apoptotic effect of silibinin. Silibinin suppressed the activation of caspase-3 by inhibiting Jun N-terminal kinase phosphorylation and the downstream hippocampal Bax/Bcl-2 ratio. Silibinin treatment significantly increased levels of synaptophysin and PSD95 in APP/PS1 transgenic mice. These results suggest that silibinin could be a potential therapeutic agent for the treatment of Alzheimer's disease.

  3. Jiaweisinisan facilitates neurogenesis in the hippocampus after stress damage

    Institute of Scientific and Technical Information of China (English)

    Lili Wu; Chuanlian Ran; Shukao Liu; Lizhen Liao; Yanling Chen; Hualei Guo; Weikang Wu; Can Yan

    2013-01-01

    The traditional Chinese medicine Jiaweisinisan has antidepressant effects, and can inhibit hypothalamus-pituitary-adrenal gland axis hyperactivity in stress-induced depression. In this study, rat hippocampal neural precursor cells were cultured in serum-free medium in vitro and a stress damage model was established with 120 μM corticosterone. Cells were treated with 10% (v/v) Jiaweisinisan drug-containing serum and the corticosterone antagonist RU38486. Results of the 3-(4,5-dimethylthiazol-2-yl)-3,5-di-phenytetrazoliumromide assay showed that both Jiaweisinisan drug-containing serum and RU38486 promoted the proliferation of neural precursor cells after corticosterone exposure. Immunofluorescence detection showed that after Jiaweisinisan drug-containing serum and RU38486 treatment, the 5-bromo-2-deoxyuridine/terminal deoxynucleotidyl transferase dUTP nick end labeling ratio in hippocampal neural precursor cells significantly increased, and the apoptotic rates of glial cells reduced, and neuron-like cell differentiation from neural precursor cells significantly increased. Our experimental findings indicate that Jiaweisinisan promotes hippocampal neurogenesis after stress damage.

  4. Caspase-3 and survivin expression in pediatric neuroblastoma and their roles in apoptosis

    Institute of Scientific and Technical Information of China (English)

    王家祥; 郑树

    2004-01-01

    Background Neuroblastoma, one of the common tumors in children, possesses the feature of natural regression that might be related to apoptosis caspase-3 and survivin are believed to respectively induce and inhibit apoptosis. We investigated the expression of caspase-3 and survivin in pediatric neuroblastoma and the role that these genes played in apoptosis.Methods The expression of caspase-3 and survivin in pediatric neuroblastoma tissue samples was detected using in situ hybridization, ter mintuesal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical staining. The role that these genes played in apoptosis was then evaluated.Results A converse correlation was observed between the expression of survivin and caspase-3. When survivin was expressed at high levels in neuroblastoma samples, caspase-3 expression was downregulated, and the apoptotic index decreased simultaneously.Conclusion There is a converse correlation between the expression of caspase-3 and the expression of survivin in neuroblastoma cells, indicating that caspase-3 might induce apoptosis, and survivin may inhibit this process.

  5. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid

    Science.gov (United States)

    Davis, Christopher G.; Eisner, Eric; McGlynn, Margaret; Shelton, John M.; Richardson, James

    2013-01-01

    Objective. To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. Design. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Results. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Conclusions. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury. PMID:26069679

  6. Effects of Spent Pot Liner on mitotic activity and nuclear DNA content in meristematic cells of Allium cepa.

    Science.gov (United States)

    Andrade-Vieira, Larissa Fonseca; de Campos, José Marcello Salabert; Davide, Lisete Chamma

    2012-09-30

    Industrial waste usually contains complex mixtures of mutagenic chemicals. Spent Pot Liner (SPL) is a complex solid waste from the aluminum industry, which is composed of organics, fluoride salts, inorganic cyanides, metals, and sodium. Due to the toxicity of these compounds, this study sought to use cytogenetics and flow cytometry to assess the effects of SPL on cell cycle parameters and DNA content in meristematic cells of Allium cepa. Three concentrations of leachates from SPL-soil mixtures were used for the study: 0, 10, and 25%. Roots were collected and analyzed after 4, 8, 12, 24, and 36 h of exposure to the above SPL leachates. The results showed an overall mitodepressive effect accompanied by an increased percentage of condensed nuclei and genomic instability as evidenced by the presence of cellular/chromosomal abnormalities. Terminal deoxynucleotidyl transferase dUTP nick end labeling revealed nuclei with fragmented DNA, a marker of programmed cell death. This study also addressed the question of reversibility of the effects of SPL and found that 36 h of exposure to 25% SPL seemed to be the point at which the effects on the induction of apoptosis became irreversible.

  7. Sestrin2 protects against acetaminophen-induced liver injury.

    Science.gov (United States)

    Kim, Seung Jung; Kim, Kyu Min; Yang, Ji Hye; Cho, Sam Seok; Kim, Ji Young; Park, Su Jung; Lee, Sang Kyu; Ku, Sae Kwang; Cho, Il Je; Ki, Sung Hwan

    2017-05-01

    Acetaminophen (APAP) overdose accounts for half of the cases of acute liver failure worldwide. We previously reported that Sestrin2 (Sesn2) protects against d-galactosamine/lipopolysaccharide-induced acute fulminant liver failure. In this study, we demonstrated that Sesn2 protects APAP-induced liver injury in mice, using a recombinant adenovirus encoding Sesn2 (Ad-Sesn2). First, we found that treatment of mice with toxic levels of APAP significantly reduced Sesn2 expression. Tail-vein injection with Ad-Sesn2 inhibited APAP-induced serum alanine aminotransferase and aspartate aminotransferase levels and markedly reduced hepatocyte degeneration and inflammatory cell infiltration. Additionally, APAP-induced glutathione depletion and reactive oxygen species generation were inhibited by Ad-Sesn2 treatment. Consistently, hepatic inflammatory gene expression and proinflammatory cytokine levels were also inhibited in Sesn2-infected mice, and we observed reduced APAP-mediated apoptotic signaling by terminal transferase-mediated dUTP nick-end labeling staining of the hepatic tissue. At a high dose of APAP, the mortality rate of Ad-Sesn2-infected mice was significantly lower than that of control mice. Furthermore, Sesn2 prevented APAP-induced damage through suppression of downstream mitogen-activated protein kinase pathway activation. Therefore, Sesn2 exerted a protective effect against APAP-induced acute liver damage by inhibiting oxidative stress and proinflammatory signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Lychee Seed Saponins Improve Cognitive Function and Prevent Neuronal Injury via Inhibiting Neuronal Apoptosis in a Rat Model of Alzheimer’s Disease

    Science.gov (United States)

    Wang, Xiuling; Wu, Jianming; Yu, Chonglin; Tang, Yong; Liu, Jian; Chen, Haixia; Jin, Bingjin; Mei, Qibing; Cao, Shousong; Qin, Dalian

    2017-01-01

    Lychee seed is a traditional Chinese medicine and possesses many activities, including hypoglycemia, liver protection, antioxidation, antivirus, and antitumor. However, its effect on neuroprotection is still unclear. The present study investigated the effects of lychee seed saponins (LSS) on neuroprotection and associated mechanisms. We established a rat model of Alzheimer’s disease (AD) by injecting Aβ25–35 into the lateral ventricle of rats and evaluated the effect of LSS on spatial learning and memory ability via the Morris water maze. Neuronal apoptosis was analyzed by hematoxylin and eosin stain and terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling analysis, and mRNA expression of caspase-3 and protein expressions of Bax and Bcl-2 by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The results showed that LSS remarkably improved cognitive function and alleviated neuronal injury by inhibiting apoptosis in the hippocampus of AD rats. Furthermore, the mRNA expression of caspase-3 and the protein expression of Bax were downregulated, while the protein expression of Bcl-2 and the ratio of Bcl-2/Bax were increased by LSS. We demonstrate that LSS significantly improves cognitive function and prevent neuronal injury in the AD rats via regulation of the apoptosis pathway. Therefore, LSS may be developed as a nutritional supplement and sold as a drug for AD prevention and/or treatment. PMID:28165366

  9. Comparison of apoptosis between adult worms of Schistosoma japonicum from susceptible (BALB/c mice) and less-susceptible (Wistar rats) hosts.

    Science.gov (United States)

    Wang, Tao; Guo, Xiaoyong; Hong, Yang; Han, Hongxiao; Cao, Xiaodan; Han, Yanhui; Zhang, Min; Wu, Miaoli; Fu, Zhiqiang; Lu, Ke; Li, Hao; Zhao, Zhixin; Lin, Jiaojiao

    2016-10-30

    Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (Pworms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing apoptosis and host interactions in schistosome infection.

  10. Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes.

    Science.gov (United States)

    Piano, Ilaria; Novelli, Elena; Della Santina, Luca; Strettoi, Enrica; Cervetto, Luigi; Gargini, Claudia

    2016-01-01

    The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis.

  11. The Extract of Aster Koraiensis Prevents Retinal Pericyte Apoptosis in Diabetic Rats and Its Active Compound, Chlorogenic Acid Inhibits AGE Formation and AGE/RAGE Interaction

    Directory of Open Access Journals (Sweden)

    Junghyun Kim

    2016-09-01

    Full Text Available Retinal capillary cell loss is a hallmark of early diabetic retinal changes. Advanced glycation end products (AGEs are believed to contribute to retinal microvascular cell loss in diabetic retinopathy. In this study, the protective effects of Aster koraiensis extract (AKE against damage to retinal vascular cells were investigated in streptozotocin (STZ-induced diabetic rats. To examine this issue further, AGE accumulation, nuclear factor-kappaB (NF-κB and inducible nitric oxide synthase (iNOS were investigated using retinal trypsin digests from streptozotocin-induced diabetic rats. In the diabetic rats, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling-positive retinal microvascular cells were markedly increased. Immunohistochemical studies revealed that AGEs were accumulated within the retinal microvascular cells, and this accumulation paralleled the activation of NF-κB and the expression of iNOS in the diabetic rats. However, AKE prevented retinal microvascular cell apoptosis through the inhibition of AGE accumulation and NF-κB activation. Moreover, to determine the active compounds of AKE, two major compounds, chlorogenic acid and 3,5-di-O-caffeoylquinic acid, were tested in an in vitro assay. Among these compounds, chlorogenic acid significantly reduced AGE formation as well as AGE/RAGE (receptor for AGEs binding activity. These results suggest that AKE, particularly chlorogenic acid, is useful in inhibiting AGE accumulation in retinal vessels and exerts a preventive effect against the injuries of diabetic retinal vascular cells.

  12. Salvianolic Acid A Attenuates Cell Apoptosis, Oxidative Stress, Akt and NF-κB Activation in Angiotensin-II Induced Murine Peritoneal Macrophages.

    Science.gov (United States)

    Li, Ling; Xu, Tongda; Du, Yinping; Pan, Defeng; Wu, Wanling; Zhu, Hong; Zhang, Yanbin; Li, Dongye

    2016-01-01

    We discuss the role of Salvianolic acid A(SAA), one of the main effective components in Salvia Miltiorrhiza (known as 'Danshen' in traditional Chinese medicine), in apoptotic factors, the production of oxidative products, and the expression of Akt and NF-κB in angiotensin II (Ang II)-mediated murine macrophages. In the present study, Ang II was added to mice abdominal macrophages with or without addition of SAA. After cell identification, apoptosis was measured by DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining, and the expression of Bcl-2 and Bax. Intracellular concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA) were also measured. Western blotting determined the expression of Akt, p-Akt, NF-κB and p-NF-κB. Ly294002 (the inhibitor of PI3K) was used to determine the mechanism of SAA. Ang II (1 µM) significantly increased the number of TUNEL-positive cells and Bax expression, but reduced Bcl-2 expression. These effects were antagonized when the cells were pretreated with SAA. SAA decreased MDA, but increased SOD in the cell lysis solution treated with Ang II. It markedly reduced the level of p-NF-κB, as also p-Akt, which was partly blocked by Ly294002. SAA prevents Ang IIinduced apoptosis, oxidative stress and related protein expression in the macrophages. It also inhibits the activation of Akt.

  13. Ulinastatin suppresses endoplasmic reticulum stress and apoptosis in the hippocampus of rats with acute paraquat poisoning

    Institute of Scientific and Technical Information of China (English)

    Hai-feng Li; Shi-xing Zhao; Bao-peng Xing; Ming-li Sun

    2015-01-01

    Lung injury is the main manifestation of paraquat poisoning. Few studies have addressed brain damage after paraquat poisoning. Ulinastatin is a protease inhibitor that can effectively stabilize lysosomal membranes, prevent cell damage, and reduce the production of free radicals. This study assumed that ulinastatin would exert these effects on brain tissues that had been poisoned with paraquat. Rat models of paraquat poisoning were intraperitoneally injected with ulinastatin. Simultaneously, rats in the control group were administered normal saline. Hematoxylin-eosin staining showed that most hippocampal cells were contracted and nucleoli had disappeared in the paraquat group. Fewer cells in the hippocampus were concentrated and nucleoli had dis-appeared in the ulinastatin group. Western blot assay showed that expressions of GRP78 and cleaved-caspase-3 were signiifcantly lower in the ulinastatin group than in the paraquat group. Immunohistochemical ifndings showed that CHOP immunoreactivity was signiifcantly lower in the ulinastatin group than in the paraquat group. Terminal deoxynucleotidyl transferase-medi-ated dUTP nick end labeling staining showed that the number of apoptotic cells was reduced in the paraquat and ulinastatin groups. These data conifrmed that endoplasmic reticular stress can be induced by acute paraquat poisoning. Ulinastatin can effectively inhibit this stress as well as cell apoptosis, thereby exerting a neuroprotective effect.

  14. Therapeutic Effects of Microbubbles Added to Combined High-Intensity Focused Ultrasound and Chemotherapy in a Pancreatic Cancer Xenograft Model

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    Yu, Mi Hye [Department of Radiology, Konkuk University Medical Center, Seoul 05030 (Korea, Republic of); Lee, Jae Young [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Kim, Hae Ri [Department of Pre-Dentistry, Gangneung-Wonju National University College of Dentistry, Gangneung 25457 (Korea, Republic of); Kim, Bo Ram; Park, Eun-Joo; Kim, Hoe Suk; Han, Joon Koo [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Choi, Byung Ihn [Department of Radiology, Chung-Ang University Hospital, Seoul 06973 (Korea, Republic of)

    2016-11-01

    To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.

  15. Functional evaluation of a novel vitreous substitute using polyethylene glycol sols injected into a foldable capsular vitreous body.

    Science.gov (United States)

    Chen, Han; Feng, Songfu; Liu, Yaqin; Huang, Zhen; Sun, Xuyuan; Zhou, Lian; Lu, Xiaohe; Gao, Qianying

    2013-09-01

    Polyethylene glycol (PEG) is a short-term (41 days) potential vitreous substitute and is too short for an ideal vitreous substitute. Previously, a foldable capsular vitreous body (FCVB) was designed to mimic vitreous function. The aim of this study is to evaluate whether PEG injected into FCVB can serve as a long-term vitreous substitute. In vitro study, a concentration of 5% (w/v) PEG sols showed natural-like mechanical and optical properties in terms of pH, density, light transmittance, refractive index, interfacial tension, viscosity, rheology, and cytotoxicity. Then in vivo tests, 30 rabbits received standard pars plana vitrectomy, of which 12 eyes were implanted with PEG injected into FCVB, nine eyes were injected with PEG sols alone, and nine others were injected with balance salt solution as control. A clinical evaluation of the anterior segment, fundus, and intraocular pressure was measured pre- and postoperatively up to 180 days, which showed that FCVBs had good retina supporting function, except for a higher incidence of cataracts. Gross pathology, hematoxylin and eosin, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining analysis also showed that FCVBs had good biocompatibility, and that all quadrants of the capsular wall fitted well with the retina. This study demonstrated that PEG injected into FCVB can serve as a long-term vitreous substitute and has potential clinical use.

  16. Histone Modification Is Involved in Okadaic Acid (OA Induced DNA Damage Response and G2-M Transition Arrest in Maize.

    Directory of Open Access Journals (Sweden)

    Hao Zhang

    Full Text Available Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA, a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph signals was altered under OA treatment. Reactive oxygen species (ROS was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

  17. Leaf-shape remodeling: programmed cell death in fistular leaves of Allium fistulosum.

    Science.gov (United States)

    Ni, Xi-Lu; Su, Hui; Zhou, Ya-fu; Wang, Feng-Hua; Liu, Wen-Zhe

    2015-03-01

    Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre-cavity cells, in the center of the leaves. Nuclei of cells within the pre-cavity site become TUNEL-positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.

  18. Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Neural apoptosis is generally believed to be mediated by two distinct pathways, caspase-dependant and caspase-independent pathways. This study investigated the apoptotic pathways involved in retinal ganglion ceils in acute diabetes in rats. Methods Diabetes was induced in male Wistar rats by a peritoneal injection of streptozotocin (STZ). Expression and localization of caspase-3 and apoptosis-inducing factor (AIF) proteins in the retina of diabetic rats was examined by Western blotting and immunohistochemistry analyses. Terminal transferase dUTP nick end labeling (TUNEL) assay and immunofluorescent staining specific for caspase-3 and AIF were applied to analyze for apoptosis of retinal ganglion cells. In addition, a caspase-3 inhibitor DEVD-CHO was injected intravitreally to further determine the apoptotic pathways of retinal ganglion cells triggered in acute diabetes. Results Two weeks after induction of diabetes, a significant increase in caspase-3 protein expression and localization occurred in the nerve fiber layer, ganglion cell layer, and inner plexiform layer of the retina. Four weeks after the onset of diabetes, the increase in caspase-3 expression was profound eight weeks postinduction of diabetes (P<0.05). Meanwhile, no AIF protein expression was detected in this study. In addition, intravitreal administration of the caspase-3 inhibitor DEVD-CHO reduced apoptosis of retinal ganglion cells by its direct inhibitory action on caspase-3. Conclusion Caspase-dependent apoptotic pathways may be the main stimulant of STZ-induced retinal ganglion cell apoptosis in acute diabetes.

  19. Fluorosis Caused Cellular Apoptosis and Oxidative Stress of Rat Kidneys

    Institute of Scientific and Technical Information of China (English)

    SONG Yang; WANG Jin-cheng; XU Hui; DU Zhen-wu; ZHANG Gui-zhen; SELIM Hamid Abdu; LI Guang-sheng

    2013-01-01

    As the strongest electronegative element,fluorine can stimulate the production of superoxide radicals in cells.In view of the important roles of kidneys in bone metabolism,the authors analyzed the quantitative pathomorphological characteristics of renal damage and the potential cellular apoptosis and oxidative stress mechanisms in rats treated with excessive fluoride.Wistar rats were exposed to 50 mg F-(110.5 mg NaF)/L,100 mg F-(221.0 mg NaF)/Land 150 mg F (331.5 mg NaF)/L in drinking water for 70 and 140 d,respectively.Microscope with image analysis was used to quantitate pathomorphological changes in renal tissues of the rats.Reactive oxygen species(ROS),the cell cycle and apoptosis of renal cells were measured by flow cytometry and TUNEL technique(terminal deoxynucleotidyl transferase dUTP nick end labeling),respectively.The ion concentrations in serum and renal functional parameters were detected by automatic biochemical analyzer.Quantitative analysis results demonstrate the expanded Bowman's space of glomerulus and obvious dilatation of renal tubule.TUNEL technique revealed that NBT/BCIP (nitro blue tetrazoliurn/5-bromo-4-chloro-3′-indolylphosphate,p-toluidine salt)-staining positive apoptotic cells selectively located in medullocortical junction areas.The data suggest that renal damage in chronic fluorostic rats is associated with the cellular apoptosis and oxidative stress.

  20. Extracellular Hb Enhances Cardiac Toxicity in Endotoxemic Guinea Pigs: Protective Role of Haptoglobin

    Directory of Open Access Journals (Sweden)

    Jin Hyen Baek

    2014-03-01

    Full Text Available Endotoxemia plays a major causative role in the myocardial injury and dysfunction associated with sepsis. Extracellular hemoglobin (Hb has been shown to enhance the pathophysiology of endotoxemia. In the present study, we examined the myocardial pathophysiology in guinea pigs infused with lipopolysaccharide (LPS, a Gram-negative bacterial endotoxin, and purified Hb. We also examined whether the administration of the Hb scavenger haptoglobin (Hp could protect against the effects observed. Here, we show that Hb infusion following LPS administration, but not either insult alone, increased myocardial iron deposition, heme oxygenase-1 expression, phagocyte activation and infiltration, as well as oxidative DNA damage and apoptosis assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL immunostaining, respectively. Co-administration of Hp significantly attenuated the myocardial events induced by the combination of LPS and Hb. These findings may have relevant therapeutic implications for the management of sepsis during concomitant disease or clinical interventions associated with the increased co-exposures to LPS and Hb, such as trauma, surgery or massive blood transfusions.

  1. Host cell apoptosis induced by infection with duck swollen head hemorrhagic disease virus

    Institute of Scientific and Technical Information of China (English)

    Chuanfeng Li; Xiaoyue Chen; Anchun Cheng; Mingshu Wang; Chanjuan Shen; Na Zhang; Yi Zhou; Dekang Zhu; Qihui Luo; Renyong Jia

    2009-01-01

    This study aimed to examine the host cell apoptosis in the tissues of Peking ducks infected with duck swollen head hemorrhagic dis-ease virus (DSHDV).The dynamic changes associated with apoptosis occurring in the internal tissues were evaluated at different time points postinoculation (PI) by performing hematoxylin and eosin (HE) staining,followed by light microscopy,terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay,and transmission electron microscopy (TEM).The results showed that DSHDV infection could induce apoptosis in host cells,including those of the bursa of Fabricius (BF),thymus,spleen,liver,intestinal tract,kidney,and esophagus.The apoptotic index (AI) values increased with time from 2 to 72 h PI,and the highest values were recorded at 72 h PI.Further,cell death due to classic necrosis was observed in the dying or deceased ducks after 72 h PI.In conclusion,host cell apoptosis can be induced by DSHDV and may play an important role in the pathogenesis of duck viral swollen head hemorrhagic disease (DVSHD).

  2. Red ginseng ameliorates acute cisplatin-induced nephropathy.

    Science.gov (United States)

    Kim, Young-Jung; Lee, Mee-Young; Son, Hwa-Young; Park, Bae-Keun; Ryu, Si-Yun; Jung, Ju-Young

    2014-06-01

    Korean red ginseng is one of the traditional herbal medicines most widely used in China, Korea, and Japan. To determine whether Korean red ginseng extract can mitigate acute renal nephropathy, we examined its renoprotective effects in a model of cisplatin-induced acute renal failure in Sprague Dawley rats. Korean red ginseng was administered to rats by oral gavage once a day at doses of 100, 300, or 500 mg/kg for 28 days. On day 23, the animals received an intraperitoneal injection of cisplatin (5 mg/kg) to induce acute renal failure. Body weight gain, urine volume, blood urea nitrogen and creatinine concentrations, and expression of p53 were measured. Terminal deoxynucleotidyl transferase dUTP nick end-labeling was used to analyze apoptosis. Kidney tissues from the control and experimental groups were analyzed by immunohistochemistry for inflammatory cytokines and histopathological examination. To identify the mechanism responsible for the renoprotective effects of Korean red ginseng, we measured malondialdehyde concentration as an end product of lipid peroxidation and the activities of the antioxidants superoxide dismutase and glutathione. Korean red ginseng significantly decreased the levels of indicators of renal dysfunction, inflammatory cytokine expression, apoptosis, and malondialdehyde content in the kidney and also significantly attenuated the histopathological changes associated with acute renal failure. These findings suggest that Korean red ginseng has renoprotective effects against cisplatin-induced acute renal failure by reducing oxidative stress and inflammation. Georg Thieme Verlag KG Stuttgart · New York.

  3. Chlorella vulgaris triggers apoptosis in hepatocarcinogenesis-induced rats

    Institute of Scientific and Technical Information of China (English)

    Emey Suhana MOHD AZAMAI; Suhaniza SULAIMAN; Shafina Hanim MOHD HABIB; Mee Lee LOOI; Srijit DAS; Nor Aini ABDUL HAMID; Wan Zurinah WANG NGAH; Yasmin Anum MOHD YUSOF

    2009-01-01

    Chlorella vulgaris (CV) has been reported to have antioxidant and anticancer properties. We evaluated the effect of CV on apoptotic regulator protein expression in liver cancer-induced rats. Male Wistar rats (200-250 g) were divided into eight groups: control group (normal diet), CDE group (choline deficient diet supplemented with ethionine in drinking water to induce hepatocarcinogenesis), CV groups with three different doses of CV (50, 150, and 300 mg/kg body weight), and CDE groups treated with different doses of CV (50, 150, and 300 mg/kg body weight). Rats were sacrificed at various weeks and liver tissues were embedded in paraffin blocks for immunohistochemistry studies. CV, at increasing doses, decreased the expression of anti-apoptotic protein, Bcl-2, but increased the expression of pro-apoptotic protein, caspase 8, in CDE rats, which was correlated with decreased hepatoctyes proliferation and increased apoptosis as determined by bromodeoxy-uridine (BrdU) labeling and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay, respectively. Our study shows that CV has definite chemopreventive effect by inducing apoptosis via decreasing the expression of Bcl-2 and increasing the expression of caspase 8 in hepatocarcinogenesis-induced rats.

  4. Ocular Lesions in Red-Tailed Hawks ( Buteo jamaicensis) With Naturally Acquired West Nile Disease.

    Science.gov (United States)

    Wünschmann, A; Armién, A G; Khatri, M; Martinez, L C; Willette, M; Glaser, A; Alvarez, J; Redig, P

    2017-03-01

    Ocular lesions are common in red-tailed hawks with West Nile (WN) disease. These lesions consist of pectenitis, choroidal or retinal inflammation, or retinal necrosis, but detailed investigation of the ocular lesions is lacking. Postmortem examination of the eyes of 16 red-tailed hawks with naturally acquired WN disease and 3 red-tailed hawks without WN disease was performed using histopathology, immunohistochemistry for West Nile virus (WNV) antigen, glial fibrillary acid protein, cleaved caspase-3, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Retinal lesions were classified as type I or type II lesions. Type I lesions were characterized by lymphoplasmacytic infiltrates in the subjacent choroid with degeneration limited to the outer retina (type Ia lesion) or with degeneration and necrosis of the outer retina or outer and inner retina (type Ib lesion) while retinal collapse, atrophy, and scarring were hallmarks of type II lesions. Type II retinal lesions were associated with a more pronounced choroiditis. Although not statistically significant, WNV antigen tended to be present in larger quantity in type Ib lesions. Type I lesions are considered acute while type II lesions are chronic. The development of retinal lesions was associated with the presence of an inflammatory infiltrate in the choroid. A breakdown of the blood-retina barrier is suspected to be the main route of infection of the retina. Within the retina, virus appeared to spread via both neuronal and Müller cell processes.

  5. Cytotoxic Effects of 2-Bromopropane on Embryonic Development in Mouse Blastocysts

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    Wen-Hsiung Chan

    2010-02-01

    Full Text Available 2-Bromopropane (2-BP, an alternative to ozone-depleting solvents, is used as a cleaning solvent. Here, we examined the cytotoxic effects of 2-bromopropane (2-BP on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Mouse blastocysts were incubated in medium with or without 2-BP (2.5, 5 or 10 μM for 24 h. Cell proliferation and growth were investigated with dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL analysis, and implantation and post-implantation development of embryos were assessed using in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 5 or 10 μM 2-BP displayed significantly increased apoptosis, and decreased inner cell mass (ICM and trophectoderm (TE cell number. Additionally, the implantation success rates of 2-BP-pretreated blastocysts were lower than those of untreated controls. In vitro treatment with 5 or 10 μM 2-BP was associated with increased resorption of postimplantation embryos, and decreased placental and fetal weights. Our results collectively indicate that in vitro exposure to 2-BP induces apoptosis, suppresses implantation rates after transfer to host mice, and retards early postimplantation development.

  6. Adequate ovarian follicular status does not prevent the decrease in pregnancy rates associated with high sperm DNA fragmentation.

    Science.gov (United States)

    Frydman, Nelly; Prisant, Nadia; Hesters, Laetitia; Frydman, René; Tachdjian, Gérard; Cohen-Bacrie, Paul; Fanchin, Rénato

    2008-01-01

    Potential reparation of sperm DNA fragmentation in the oocyte may disturb any relationship between DNA-damaged sperm and the implantation ability of resulting embryos. To rule out this factor, we analyzed the consequences of sperm DNA fragmentation on IVF-ET outcome in women with healthy ovarian function. Prospective study. Teaching hospital, France. All 117 women were <38 years old, who combined normal serum day-3 FSH and inhibin B levels with an adequate response to controlled ovarian hyperstimulation. The DNA fragmentation rate was determined in the raw sperm used for conventional IVF by flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Cycles were sorted into two groups according to whether DNA fragmentation exceeded (high fragmentation [HF], n = 52) or did not exceed (low fragmentation [LF], n = 65) the 50th percentile of values (35%). D2 embryo quality and implantation and ongoing pregnancy rates. Patients' characteristics, raw semen parameters, fertilization rates, and embryology data were similar in HF and LF groups. Clinical (37.5% vs. 62.5%) and ongoing (23.5% vs. 57.8%) pregnancy rates per ET and implantation rates (24.5% vs. 42.4%) were lower in the HF group than in the LF group. High sperm DNA fragmentation spares fertilization and top embryo morphology rates but is associated with decreased IVF-ET outcome.

  7. Crude extract of Fusarium oxysporum induces apoptosis and structural alterations in the skin of healthy rats.

    Science.gov (United States)

    de Paulo, Luis F; Coelho, Ana C; Svidzinski, Terezinha I E; Sato, Francielle; Rohling, Jurandir H; Natali, Maria Raquel M; Baesso, Mauro L; Hernandes, Luzmarina

    2013-09-01

    We evaluate the biological and physicochemical effects of a Fusarium oxysporum crude extract (CE) on the skin of healthy rats. The CE is topically applied and subsequently the skin is collected after 3, 6, 12, and 24 h. The samples are analyzed by Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) and histomorphometric analysis. Terminal dUTP nick end labeling (TUNEL assay) is performed to detect both the cells in apoptosis and proliferation. There is a thickening of the epidermis after 6, 12, and 24 h and dermis after 12 and 24 h of CE application. A reduction of the dermis thickness is observed at 3 and 6 h. The treated skin shows higher labeling intensity by TUNEL at 3 h, while a higher intensity by proliferating cell nuclear antigen occurs at 3 and 12 h. FTIR-PAS data support the histology observations showing an increase in the absorption peaks in the dermis after the application of the CE. F. oxysporum CE permeated through the epidermis and the dermis, reaching the subcutaneous tissue, inducing cell apoptosis, and causing physicochemical changes in the organic molecules located in the dermis. This is the first known study associating histopathological and physical chemistry changes on healthy skin after the application of F. oxysporum CE.

  8. p53 and telomerase control rat myocardial tissue response to hypoxia and ageing

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    A. Cataldi

    2009-12-01

    Full Text Available Cellular senescence implies loss of proliferative and tissue regenerative capability. Also hypoxia, producing Reactive Oxygen Species (ROS, can damage cellular components through the oxidation of DNA, proteins and lipids, thus influencing the shortening of telomeres. Since ribonucleoprotein Telomerase (TERT, catalyzing the replication of the ends of eukaryotic chromosomes, promotes cardiac muscle cell proliferation, hypertrophy and survival, here we investigated its role in the events regulating apoptosis occurrence and life span in hearts deriving from young and old rats exposed to hypoxia. TUNEL (terminal-deoxinucleotidyl -transferase- mediated dUTP nick end-labeling analysis reveals an increased apoptotic cell number in both samples after hypoxia exposure, mainly in the young with respect to the old. TERT expression lowers either in the hypoxic young, either in the old in both experimental conditions, with respect to the normoxic young. These events are paralleled by p53 and HIF-1 ? expression dramatic increase and by p53/ HIF-1 ? co-immunoprecipitation in the hypoxic young, evidencing the young subject as the most stressed by such challenge. These effects could be explained by induction of damage to genomic DNA by ROS that accelerates cell senescence through p53 activation. Moreover, by preventing TERT enzyme down-regulation, cell cycle exit and apoptosis occurrence could be delayed and new possibilities for intervention against cell ageing and hypoxia could be opened.

  9. Ischemic Postconditioning-Regulated miR-499 Protects the Rat Heart Against Ischemia/Reperfusion Injury by Inhibiting Apoptosis through PDCD4

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    Jianbing Zhu

    2016-11-01

    Full Text Available Background: Here, we determined miR-499 involvement in the protective effect of ischemic postconditioning (IPC against myocardial ischemia/reperfusion (I/R injury and identified the underlying mechanisms. Methods: To investigate the cardioprotective effect of IPC-induced miR-499, rats were divided into the following five groups: sham, I/R, IPC, IPC + scramble, and IPC + antagomiR-499. Hemodynamic indexes were measured by carotid-artery intubation to assess left ventricular function . Ischemia and infarction areas of rat hearts were determined by Evans blue and triphenyltetrazolium chloride staining, and cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end-labeling assay. Results: IPC attenuated I/R-induced infarct size of the left ventricle (45.28 ± 5.40% vs. 23.56 ± 6.20%, P vs. 990.21 ± 172.39%, P vs. 1289.11 ± 347.28%, P vs. 4.85 ± 1.52%, P in vivo and in vitro by knockdown of cardiac miR-499, suggesting that miR-499 may participate in the protective function of IPC against I/R injury through targeting programmed cell death 4 (PDCD4. Conclusion: Our data revealed that IPC-regulated miR-499 plays an important role in IPC-mediated cardiac protection against I/R injury by targeting PDCD4.

  10. Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition

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    D.Y. Xia

    2013-08-01

    Full Text Available Sublethal ischemic preconditioning (IPC is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration, 5×5 min (5 min duration, 2 episodes, 15-min interval, 5×5×5 min (5 min duration, 3 episodes, 15-min intervals, and 15 min (15 min duration, and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.

  11. Arsenic trioxide treatment of rabbit liver VX-2 carcinoma via hepatic arterial cannulation-induced apoptosis and decreased levels of survivin in the tumor tissue.

    Science.gov (United States)

    Li, Hong; Gong, Jian; Jiang, Xuyuan; Shao, Haibo

    2013-02-01

    To investigate the role of tumor apoptosis-inhibitory protein survivin in arsenic trioxide-induced apoptosis in VX-2 carcinoma in the rabbit liver by means of transcatheter arterial chemoembolization. Sixteen rabbits with 32 implanted hepatic VX-2 tumors were randomly divided into two groups. The experimental group received 2 mg of arsenic trioxide and 1 mL of ultra-fluid lipiodol co-injected via hepatic arterial cannulation and the control group received only 1 mL of lipiodol. Animals were sacrificed 3 weeks after trans-catheterial arterial chemoembolization. Tumor tissue and tumor-peripheral tissue were collected for analysis. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling staining was used to assess tumor cells apoptosis. Immunohistochemistry was used to assess the presence of survivin protein. Reverse transcription polymerase chain reaction was used to determine the expression of survivin gene. The number of apoptotic cells significantly increased in the tumor tissue (5.20 ± 0.60%) compared to tumor-peripheral tissue (1.29 ± 0.42%) of the arsenic trioxide-treated group. Survivin expression levels in the tumor tissue were significantly reduced in arsenic trioxide-treated group (7.68 ± 0.65) compared to the control group (35.30 ± 4.63). Transcatheter arterial chemoembolization with arsenic trioxide induced apoptosis of VX-2 carcinoma, in which tumor apoptosis-inhibitory protein survivin may have played a role.

  12. Occurrence of complement protein C3 in dying pyramidal neurons in rat hippocampus after systemic administration of kainic acid.

    Science.gov (United States)

    Morita, Hiroyuki; Suzuki, Katsuaki; Mori, Norio; Yasuhara, Osamu

    2006-11-27

    To evaluate the roles of complement in kainic acid (KA)-induced neuronal damages, the immunohistochemical localization of the complement protein C3 was examined in rat hippocampus after systemic KA injection. The immunoreactivity for C3 was found in glial cells in control rats, and such glial cells were increased in number after KA injection. Our confocal study showed that C3-positive glial cells were microglia. Three to seven days after KA, C3 immunoreactivity appeared in CA1 and CA3 pyramidal neurons. Double staining for C3 combined with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling showed that occurrence of C3 immunoreactivity in neurons coincided well with that of DNA fragmentation. Western blot analysis and RT-PCR experiments suggested local synthesis of C3 by brain cells. Our results suggest that C3 contributes greatly to neuronal death after systemic KA administration, and that microglia and neurons are the local source of C3 in KA-induced brain injury.

  13. Effects of sargentgloryvine stem extracts on HepG-2 cells in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Ming-Hua Wang; Min Long; Bao-Yi Zhu; Shu-Hui Yang; Ji-Hong Ren; Hui-Zhong Zhang

    2011-01-01

    AIM: To observe the effects of sargentgloryvine stem extracts (SSE) on the hepatoma cell line HepG-2 in vitro and in vivo and determine its mechanisms of action.METHODS: Cultured HepG-2 cells treated with SSE were analysed by 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-Diphenyltetrazolium bromide and clone formation assay.The cell cycle and apoptosis analysis were conducted by flow cytometric, TdT-Mediated dUTP Nick End Labeling and acridine orange/ethidium bromide staining methods,and protein expression was examined by both reverse transcriptase-polymerase chain reaction and Western blotting.The pathological changes of the tumor cells were observed by haematoxylin and eosin staining. Tumor growth inhibition and side effects were determined in a xenograft mouse model.RESULTS: SSE treatment could not only inhibit HepG-2 cell proliferation in a dose- and time-dependent manner but also induce apoptosis and cell cycle arrest at the S phase. The number of colonies formed by SSEtreated tumor cells was fewer than that of the controls (P 0.05). Systemic administration of SSE could inhibit the HepG-2 xenograft tumor growth with no obvious toxic side effects on normal tissues.CONCLUSION: SSE can induce apoptosis of HepG-2 cells in vitro and in vivo through decreasing expression of Bcl-xl and Mcl-1 and increasing expression of Bax.

  14. Korean Red Ginseng water extract inhibits COX-2 expression by suppressing p38 in acrolein-treated human endothelial cells

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    Seung Eun Lee

    2014-01-01

    Full Text Available Cigarette smoke is considered a major risk factor for vascular diseases. There are many toxic compounds in cigarette smoke, including acrolein and other α,β-unsaturated aldehydes, which are regarded as mediators of inflammation and vascular dysfunction. Furthermore, recent studies have revealed that acrolein, an α,β-unsaturated aldehyde in cigarette smoke, induces inflammatory mediator expression, which is known to be related to vascular diseases. In this study, we investigated whether Korean Red Ginseng (KRG water extract suppressed acrolein-induced cyclooxygenase (COX-2 expression in human umbilical vein endothelial cells (HUVECs. Acrolein-induced COX-2 expression was accompanied by increased levels of phosphorylated p38 in HUVECs and KRG inhibited COX-2 expression in HUVECs. These results suggest that KRG suppresses acrolein-induced COX-2 expression via inhibition of the p38 mitogen-activated protein kinase signaling pathway. In addition, KRG exhibited an inhibitory effect on acrolein-induced apoptosis, as demonstrated by annexin V–propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Consistent with these results, KRG may exert a vasculoprotective effect through inhibition of COX-2 expression in acrolein-stimulated human endothelial cells.

  15. A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells.

    Science.gov (United States)

    Jung, Il Lae; Lee, Ju Hye; Kang, Se Chan

    2015-09-01

    It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44-52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers.

  16. Caffeine-induced nuclear translocation of FoxO1 triggers Bim-mediated apoptosis in human glioblastoma cells.

    Science.gov (United States)

    Sun, Fei; Han, Dong-Feng; Cao, Bo-Qiang; Wang, Bo; Dong, Nan; Jiang, De-Hua

    2016-03-01

    Caffeine is one of the most commonly ingested neuroactive compounds and exhibits anticancer effects through induction of apoptosis and suppression of cell proliferation. However, the mechanisms underlying these effects are currently unknown. In this study, we investigated the mechanisms of caffeine-induced apoptosis in U251 cells (human glioma cell line). We analyzed the inhibitory effects of caffeine on cell proliferation by performing WST-8 and colony formation assays; in addition, cell survival was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and flow cytometric analysis. Western blotting was used to investigate the role played by FoxO1 in the proapoptotic effects of caffeine on glioma cells. Results showed that caffeine inhibited proliferation and survival of human glioma cells, induced apoptosis, and increased the expression of FoxO1 and its proapoptotic target Bim. In addition, we found that FoxO1 enhanced the transcription of its proapoptotic target Bim. In summary, our data indicates that FoxO1-Bim mediates caffeine-induced regression of glioma growth by activating cell apoptosis, thereby providing new mechanistic insight into the possible use of caffeine in treating human cancer.

  17. Anticancer Activities of Trichostatin A on Maligant Lymphoid Cells

    Institute of Scientific and Technical Information of China (English)

    SUN Chunyan; LIU Xinyue; CHEN Yan; LIU Fang

    2006-01-01

    The anticancer activity of trichostain A (TSA) on human B cell non-Hodgkin's lymphoma and its mechanism were explored. The effect of TSA on the growth of Raji cells and normal peripheral blood mononuclear cells (NPBMNC) was studied by MTT assay. The effect of TSA on the apoptosis of Raji cells and NPBMNC was studied by flow cytometry and TDT-mediated dUTP nick end labeling (TUNEL). The effect of TSA on the cell cycle of Raji cells was studied by propidium iodide method. The results showed that TSA potently inhibited proliferation of Raji cells at microgram concentrations and induced apoptosis of Raji cells in a time- and concentration-dependent manner.Treatment with TSA induced accumulation of cells in G0/G1 or G2/M and a concomitant decrease of cell population in S phase. However, NPBMNC was less sensitive to the cytotoxic effect of TSA than Raji cells. It was concluded that TSA may inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Moreover, TSA demonstrates low toxicity in NPBMNC but selectively induces apoptosis of Raji cells.

  18. Edaravone inhibits apoptosis caused by ischemia/reperfusion injury in a porcine hepatectomy model

    Institute of Scientific and Technical Information of China (English)

    Mitsugi Shimoda; Yoshimi Iwasaki; Toshie Okada; Keiichi Kubota

    2012-01-01

    AIM:To investigate the effect of E3-methyl-1-phenyl-2-pyrazolin-5-one (Edr) on hepatic ischemia-reperfusion (I/R) injury and liver regeneration in a porcine hepatectomy model.METHODS:One hour ischemia was induced by occluding the vessels and the bile duct of the right and median lobes.A 40% left hepatectomy was performed after reperfusion.Six animals received Edr (3 mg/kg per hour)intravenously and six control animals received saline just before reperfusion.Remnant liver volume,hemodynamics,aspartate aminotransferase (AST),alanine aminotransferase,lactate dehydrogenase and lactic acid,were compared between the groups.The expression of transforming growth factor-β (TGF-β1) and toll-like receptor (TRL) mRNA in hepatic tissues was examined using reverse transcription polymerase chain reaction.Apoptosis was demonstrated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining,respectively.RESULTS:Serum AST (P =0.029),and toll like receptor 4 level (P =0.043) were significantly lower after 3 hin animals receiving Edr.In addition,TUNEL staining in Edr-treated pigs showed significantly fewer hepatocytes undergoing apoptosis compared with control pigs.After mo,all factors were non-significantly different between the two groups.CONCLUSION:Edr is considered to reduce hepatic injury in the early stage of I/R injury in a porcine model.

  19. Anticancer effects of oligomeric proanthocyanidins on human colorectal cancer cell line, SNU-C4

    Institute of Scientific and Technical Information of China (English)

    Youn-Jung Kim; Hae-Jeong Park; Seo-Hyun Yoon; Mi-Ja Kim; Kang-Hyun Leem; Joo-Ho Chung; Hye-Kyung Kim

    2005-01-01

    AIM: Oligomeric proanthocyanidins (OPC), natural polyphenolic compounds found in plants, are known to have antioxidant and anti-cancer effects. We investigated whether the anti-cancer effects of the OPC are induced by apoptosis on human colorectal cancer cell line, SNU-C4.METHODS: Colorectal cancer cell line, SNU-C4 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cytotoxic effect of OPC was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt-etrazolium bromide (MTT) assay. To find out the apoptotic cell death, 4, 6-diamidino-2-phenylindole (DAPI) staining,terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcriptionpolymerase chain reaction (RT-PCR), and caspase-3 enzyme assay were performed.RESULTS: In this study, cytotoxic effect of OPC on SNUC4 cells appeared in a dose-dependent manner. OPC treatment (100 μg/mL) revealed typical morphological apoptotic features. Additionally OPC treatment (100 μg/mL)increased level of BAX and CASPASE-3, and decreased level of BCL-2 mRNA expression. Caspase-3 enzyme activity was also significantly increased by treatment of OPC (100 μg/mL) compared with control.CONCLUSION: These data indicate that OPC caused cell death by apoptosis through caspase pathways on human colorectal cancer cell line, SNU-C4.

  20. Apoptosis of transgenic cloned and recloned bovine blastocysts

    Institute of Scientific and Technical Information of China (English)

    Guojie Sun; Rong Li; Yunping Dai; Haiping Wang; Lili Wang; Ying Liu; Fangrong Ding; Hengxi Wei; Ning Li

    2009-01-01

    Apoptosis plays an important role in preimplantation embryonic development. Investigating mechanisms of apoptosis can provide useful information for obtaining high-quality embryos and help to improve cloning efficiency. Here, we investigated the incidence of blastomere apoptosis in transgenic blastocysts generated by somatic cell nuclear transfer (SCNT) and recloning using a terminal deoxy-nucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. Transgenic recloned embryos were the second generation SCNT embryos derived from the somatic cells of a transgenic SCNT calf. The blastocyst rate of transgenic SCNT embryos was lower than that of nontransgenic SCNT embryos. The incidence of apoptosis in transgenic SCNT embryos was higher than that of nontrans-genie SCNT embryos. The blastocyst rate and the incidence of apoptosis in transgenic recloned embryos were similar to nontransgenic SCNT embryos. The process of donor cell transfection and drug selection may decrease the developmental capacity of transgenic SCNT embryos. Serial cloning did not influence the developmental capacity of transgenic recloned embryos.

  1. Patterns of Apoptosis and Proliferation throughout the Biennial Reproductive Cycle of Viviparous Female Typhlonectes compressicauda (Amphibia, Gymnophiona

    Directory of Open Access Journals (Sweden)

    Michel Raquet

    2016-12-01

    Full Text Available Typhlonectes compressicauda is an aquatic gymnophionan amphibian living in South America. Its breeding cycle is linked to seasons, characterized by a regular alternation of rainy and dry seasons. During a complex biennial cycle, the female genital tract undergoes a series of alternations of increasing and decreasing, governed by equilibrium of proliferation and apoptotic phenomena. Immunohistochemical methods were used to visualize cell proliferation with the detection of Ki67 antibody, a protein present in proliferative cells; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL and Apostain were performed to detect apoptotic cells on sections of ovaries and oviducts. In ovaries, both phenomena affect the germinal nests and follicles according to the cycle period. In the oviduct, the balance was in favor of proliferation during preparation for reproduction, and in favor of apoptosis when genital ducts regress. Apoptosis and proliferation are narrowly implicated in the remodeling of the genital tract and they are accompanied by the differentiation of tissues according to the phase of the breeding cycle. These variations permit the capture of oocytes at ovulation, always at the same period, and the parturition after 6–7 months of gestation, at a period in which the newborns live with their mother, protected in burrows in the mud. During the intervening year of sexual inactivity, the female reconstitutes body reserves.

  2. Continuous intraperitoneal carboplatin delivery for the treatment of late-stage ovarian cancer.

    Science.gov (United States)

    Zhidkov, Nickholas; De Souza, Raquel; Ghassemi, Amir H; Allen, Christine; Piquette-Miller, Micheline

    2013-09-01

    The rate of failure of chemotherapy treatment in ovarian cancer remains high, resulting in a low 5-year survival rate of 20-40% in patients that present with advanced-stage disease. Treatment-free periods between cycles of chemotherapy may contribute to accelerated tumor cell proliferation and decreased treatment response. The elimination of treatment-free breaks has been deemed beneficial in the context of cell-cycle-specific agents. The potential benefit of this approach for non-cell-cycle-specific agents has not yet been elucidated. The present study is the first to address this issue by investigating the impact of continuous versus intermittent intraperitoneal administration of carboplatin over a 14 day period to SCID mice bearing SKOV-3 ovarian cancer xenografts. Immunostaining of tumor sections was employed to quantify tumor proliferation, angiogenesis, and apoptosis using Ki-67, CD-31, caspase-3 (CASP3), and terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). Continuous ip administration of carboplatin resulted in greater tumor growth inhibition than intermittent therapy (p < 0.05). Significantly greater tumor cell apoptosis and less cell proliferation and angiogenesis were measured in tumors of mice treated with continuous carboplatin as compared to both intermittent and control groups. These results indicate that continuous local administration may be a promising approach to improve the effectiveness of platinum-based chemotherapy regimens.

  3. Effect of Tindurin on Immunopathogenesis Mechanism of Collagen-Induced Arthritis

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    F Saadat

    2005-08-01

    Full Text Available Rheumatoid arthritis is a chronic inflammatory disease characterized by the sequestration of various leukocyte subpopulations within both the developing pannus and synovial space. This study was undertaken to examine the therapeutic potency of tindurin in experimental rheumatoid arthritis. Collagen-induced arthritis (CIA was induced by intradermally immunization of Lewis rats at the base of the tail. The paws and knees were then removed for histopathology and radiography analysis. Using fibrosarcoma cell line the apoptosis process was measured by Terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL method. Our data showed that the i.p. injection of tindurin to arthritic rats induced a significant reduction in paw edema. Histopathological assessment showed reduced inflammatory cells infiltrate, tissue edema and bone erosion in joints of treated rats. Moreover, our results in radiography were in line with histological findings as well as tindurin was found to induce apoptosis of treated cells in comparison with positive, negative and non-treated ones. Our findings revealed the therapeutic effect of tindurin in experimental model of rheumatoid arthritis in comparison with methotrexate as a choice drug.

  4. Paeoniflorin ameliorates acute necrotizing pancreatitis and pancreatitis‑induced acute renal injury.

    Science.gov (United States)

    Wang, Peng; Wang, Weixing; Shi, Qiao; Zhao, Liang; Mei, Fangchao; Li, Chen; Zuo, Teng; He, Xiaobo

    2016-08-01

    Acute renal injury caused by acute necrotizing pancreatitis (ANP) is a common complication that is associated with a high rate of mortality. Paeoniflorin is the active ingredient of paeonia radix and exhibits a number of pharmacological effects, such as anti‑inflammatory, anticancer, analgesic and immunomodulatory effects. The present study detected the potential treatment effects of paeoniflorin on acute renal injury induced by ANP in a rat model. The optimal dose of paeoniflorin for preventing acute renal injury induced by ANP was determined. Then, the possible protective mechanism of paeoniflorin was investigated. The serum levels of tumor necrosis factor (TNF)‑α, interleukin (IL)‑1β and IL‑6 were measured with enzyme‑linked immunosorbent assay kits. Renal inflammation and apoptosis were measured by immunohistochemistry and terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling assay. The expression of nitric oxide in kidney tissues was also evaluated. The p38 mitogen‑activated protein kinases (MAPKs) were measured by western blotting. The results shown that paeoniflorin may ameliorate acute renal injury following ANP in rats by inhibiting inflammatory responses and renal cell apoptosis. These effects may be associated with the p38MAPK and nuclear factor‑κB signal pathway.

  5. pSVPoMcat modifying Schwann cell to protect injured spinal neurons in rats

    Institute of Scientific and Technical Information of China (English)

    陈礼刚; 高立达; 朴永旭; 毛伯镛; 曾凡俊

    2002-01-01

    Objective: To investigate the protective effect of pSVPoMcat (myelin basic protein microgene)modifying Schwann cell on injured spinal neurons.Methods: A model of rat spinal cord injured by hemisection was used. One hundred and twenty healthy SD rats of both sexes weighing 250-300 g were divided into three groups: Group A (n=40, treated with implantation of pSVPoMcat modifying Schwann cell), Group B (n= 40, treated with implantation of Schwann cell only) and Group C (n=400, treated with sham operation as the control). One week after operation the rat functional recovery was observed dynamically by using combined behavioral score (CBS) and cortical somatasensory evoked potentials, the spinal cord sections were stained by Nissl, acid phosphatase enzyme histochemistry and cell apoptosis was examined by methye green, terminal deoxynucleotidyl and the dUTP Nick end labeling technique. Quantitative analysis was done by computer image analysis system.Results: In Group A the injured neurons recovered well morphologically. The imaging analysis showed a result of Group A>Group B>Group C in the size of the neurons (P<0.01). The percentage of ACP (acid phosphatase) stained area and the rate of apoptosis sequence were groups A

  6. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

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    Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)

    2013-10-15

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  7. Phytophthora sojae TatD nuclease positively regulates sporulation and negatively regulates pathogenesis.

    Science.gov (United States)

    Chen, Linlin; Shen, Danyu; Sun, Nannan; Xu, Jing; Wang, Wen; Dou, Daolong

    2014-10-01

    During pathogenic interactions, both the host and pathogen are exposed to conditions that induce programmed cell death (PCD). Certain aspects of PCD have been recently examined in eukaryotic microbes but not in oomycetes. Here, we identified conserved TatD proteins in Phytophthora sojae; the proteins are key components of DNA degradation in apoptosis. We selected PsTatD4 for further investigation because the enzyme is unique to the oomycete branch of the phylogenetic tree. The purified protein exhibited DNase activity in vitro. Its expression was upregulated in sporangia and later infective stages but downregulated in cysts and during early infection. Functional analysis revealed that the gene was required for sporulation and zoospore production, and the expression levels were associated with the numbers of hydrogen-peroxide-induced terminal dUTP nick end-labeling-positive cells. Furthermore, overexpression of PsTatD4 gene reduced the virulence in a susceptible soybean cultivar. Together, these data suggest that apoptosis may play different roles in the early and late infective stages of P. sojae, and that PsTatD4 is a key regulator of infection. The association of PsTatD4 and apoptosis will lay a foundation to understanding the basic biology of apoptosis and its roles in P. sojae disease cycle.

  8. Prevention effects of Schisandra polysaccharide on radiation-induced immune system dysfunction.

    Science.gov (United States)

    Zhao, Lian-Mei; Jia, Yun-Long; Ma, Ming; Duan, Yu-Qing; Liu, Li-Hua

    2015-05-01

    In this study, we investigate the efficacy of SP (Schisandra polysaccharide) in prevention of radiation-induced immune dysfunction and discussed the underlying mechanisms with a Bal/bc mouse model. The data demonstrated that SP could reverse the decreases in the number of white blood cells and lymphocytes in peripheral blood. In addition, the immunoglobulin G (IgG) and complement C3 in blood serum were all decreased after radiation and SP could restore this radiation disorder. Furthermore, SP could reverse the deregulation of CD3(+)CD4(+) and CD3(+)CD8(+) T cell subsets in peripheral blood and thymus of mice after radiotherapy. We also performed terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and Immunohistochemistry (IHC) to investigate the apoptosis and underlying mechanisms of SP in thymus. Data showed that radiation-induced apoptosis of thymocytes could be reversed by SP through inducing upregulation of Bcl-2 expression and downregulation of Fas and Bax levels. Furthermore, SP has no any side-effects on immunity of normal mice. In conclusion, our results indicated that SP could effectively prevent immune injury during radiotherapy by protecting the immune system. This valuable information should be of assistance in choosing a rational design for therapeutic interventions of prevention immune system damage in the radiation treatment.

  9. Effects of fasting and refeeding on intestinal cell proliferation and apoptosis in hammerhead shark (Sphyrna lewini)

    Institute of Scientific and Technical Information of China (English)

    Hideya Takahashi; Susumu Hyodo; Tsukasa Abe; Chiyo Takagi; Gordon E Grau; Tatsuya Sakamoto

    2014-01-01

    Objective: To examine the effects of fasting and refeeding on intestinal cell proliferation and apoptosis in an opportunistic predator, hammerhead shark (Sphyrna lewini) of elasmobranch fishes which are among the earliest known extant groups of vertebrates to have the valvular intestine typical for the primitive species.Methods:5-day refeeding. Intestinal apoptosis and cell proliferation were assessed by using oligonucleotide detection assay, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry of proliferating cells nuclear antigen.Results:Animals were euthanized after 5-10 d of fasting or feeding, or after 10-day fasting and generally decreased during fasting. Numerous apoptotic cells were observed around the tips of the villi, primarily in the epithelium in the fed sharks, whereas fewer labeled nuclei were detected in the epithelium of fasted sharks. Refeeding returned intestinal apoptosis to the level in the fed sharks. Proliferating cells were observed in the epithelium around the troughs of the villi and greater in number in fed sharks, whereas fewer labeled nuclei were detected in fasted sharks. Plasma levels of cholesterol and glucose were reduced by fasting. Intestinal apoptosis Conclusions: The cell turnover is modified in both intestinal epithelia of the shark and the murines by fasting/feeding, but in opposite directions. The difference may reflect the feeding ecology of the elasmobranchs, primitive intermittent feeders.

  10. Establishment of a blue light damage model of human retinal pigment epithelial cells in vitro.

    Science.gov (United States)

    Su, G; Cai, S J; Gong, X; Wang, L L; Li, H H; Wang, L M

    2016-06-24

    To establish a blue-light damage model of human retinal pigment epithelium (RPE). Fourth-generation human RPE cells were randomly divided into two groups. In group A, cells were exposed to blue light (2000 ± 500 lux) for 0 (control), 3, 6, 9, and 12 h, and cell culture was stopped after 12 h. In group B, cells were exposed to blue light at the same intensity and time periods, but cell culture was stopped after 24 h. TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed to determine the most suitable illuminating time with apoptotic index. Flow cytometry was used to determine apoptotic ratio of RPEs. In group A, the apoptotic index of cells that received 6, 9 and 12 h of blue light was higher than that of control. The apoptotic index of cells receiving 9 and 12 h was higher than that of 6 h (P = 0.000). In group B, the apoptotic index and RPE cell apoptosis ratio of cells exposed to 6, 9 and 12 h of blue light were higher than that of 3 h (P = 0.000); and cells receiving 9 and 12 h had higher values than that of 6 h. This study demonstrated that the best conditions to establish a blue light damage model of human retinal pigment epithelial cells in vitro are 2000 ± 500 lux light intensity for 6 h, with 24 h of cell culture post-exposure.

  11. Postnatal development of hypoplastic thymus in semi-lethal dwarf pet/pet males.

    Science.gov (United States)

    Chiba, Junko; Suzuki, Hiroetsu; Aoyama, Hiroaki; Katayama, Kentaro; Suzuki, Katsushi

    2011-04-01

    The petit rat (pet/pet) is a new semi-lethal dwarf mutant with anomalies in the thymus and testes, defects inherited as a single autosomal recessive trait. At birth, these pet/pet rats show low birth weight and extremely small thymuses; at 140 days of age, their thymuses show abnormal involution. In the present study, we examined early postnatal development of hypoplastic pet/pet thymuses. In addition to being hypoplastic at birth, pet/pet thymus growth was almost completely impaired during the early postnatal period. As shown by cellular incorporation of BrdU, the mitotic activity was lower in pet/pet than in normal thymuses, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that apoptosis occurred more often in pet/pet than in normal thymus cells during the first few days after birth. These results indicate that postnatal development of the hypoplastic pet/pet thymus is defective due to the reduced proliferation and increased apoptosis of thymic cells.

  12. Developmental changes in cell proliferation and apoptosis in the normal duck thymus.

    Science.gov (United States)

    Fang, J; Cui, H; Peng, X; Chen, Z; He, M; Tang, L

    2011-12-01

    Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.

  13. Profile of hepatocyte apoptosis and bile lakes before and after bile duct decompression in severe obstructive jaundice patients

    Institute of Scientific and Technical Information of China (English)

    ToarJMLalisang; RadenSjamsuhidajat; NurjatiCSiregar; AkmalTaher

    2010-01-01

    BACKGROUND: Excessive hepatocyte apoptosis and bile lakes in severe obstructive jaundice might impair liver functions. Although decompression of the bile duct has been reported to improve liver functions in animal studies, the mechanism of obstruction differs from that in humans. This study aimed to determine the profiles of hepatocyte apoptosis and bile lakes following bile duct decompression in patients with severe obstructive jaundice in the clinical setting. METHODS: We conducted a "before and after study" on severe obstructive jaundice patients as a model of inhibition of the excessive process by bile duct decompression. Specimens of liver biopsies were taken before and after decompression of the bile duct and then stained by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) to identify hepatocyte apoptosis and by hematoxilin-eosin (HE) to identify bile lakes. All measurements were independently done by 2 observers. RESULTS: Twenty-one severe obstructive jaundice patients were included. In all patients, excessive hepatocyte apoptosis and bile lakes were apparent. After decompression, the hepatocyte apoptosis index decreased from 53.1 (SD 105) to 11.7 (SD 13.6) (P CONCLUSION: Bile duct decompression improves hepatocyte apoptosis and bile lakes in cases of severe obstructive jaundice, similar to the findings in animal studies.

  14. Mizoribine ameliorates renal injury and hypertension along with the attenuation of renal caspase-1 expression in aldosterone-salt-treated rats.

    Directory of Open Access Journals (Sweden)

    Toshiki Doi

    Full Text Available Aldosterone-salt treatment induces not only hypertension but also extensive inflammation that contributes to fibrosis in the rat kidney. However, the mechanism underlying aldosterone-salt-induced renal inflammation remains unclear. Pyroptosis has recently been identified as a new type of cell death that is accompanied by the activation of inflammatory cytokines. We hypothesized that aldosterone-salt treatment could induce inflammation through pyroptosis and that mizoribine, an effective immunosuppressant, would ameliorate the renal inflammation that would otherwise cause renal fibrosis. Ten days after recovery from left uninephrectomy, rats were given drinking water with 1% sodium chloride. The animals were divided into three groups (n = 7 per group: (1 vehicle infusion group, (2 aldosterone infusion group, or (3 aldosterone infusion plus oral mizoribine group. Aldosterone-salt treatment increased the expression of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 and caspase-1, and also increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. However, the oral administration of mizoribine attenuated these alterations. Furthermore, mizoribine inhibited hypertension and renal fibrosis, and also attenuated the aldosterone-induced expression of serum/glucocorticoid-regulated kinase and α epithelial sodium channel. These results suggest that caspase-1 activation plays an important role in the development of inflammation induced by aldosterone-salt treatment and that it functions as an anti-inflammatory strategy that protects against renal injury and hypertension.

  15. NK/T细胞淋巴瘤细胞凋亡和细胞增殖特征及意义%The significance and features of apoptosis and proliferation of NK/T cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    Dabin Wang; Meng Ming; Junhua Liu; Jianhua Yi; Dianding Zou

    2011-01-01

    Objective:The aim was to study the features and clinical significance of cell apoptosis and proliferation of NK/T cell lymphoma. Methods:TdT-mediated dUTP nick end labeling and immunohistochemical Streptavidin-peroxidase method were used to study cell apoptosis and the expression of proliferation cell nuclear antigen in 25 NK/T cell lymphoma and 10reactive lymphoid tissues. Results:Apoptotic index (AI) and proliferative index (PI) averaged (1.92% ± 0.86%) and (41.48%± 5.10%) respectively in the 25 NK/T cell lymphomas and (6.70% ± 1.89%) and (20.10% ± 2.77%) in the 10 reactive lymphoid tissues. Compared with reactive lymphoid tissues, AI was significantly reduced in NK/T cell lymphoma (t = 10.80, P < 0.01)while PI significantly increased (t = 12.39, P < 0.01). In addition, in NK/T cell lymphoma, AI and PI were positively related (r = 0.69, P < 0.01). Conclusion:In NK/T cell lymphoma, cell apoptosis is reduced while cell proliferation increased. The imbalance between cell apoptosis and cell proliferation is closely related to the development and progression of NK/T cell lymphoma.

  16. Detection of apoptotic cells and immunohistochemical study of bcl-2 and p53 gene protein in primary gastric mucosa-associated lymphoid tissue (MALT) lymphoma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To identify the apoptotic cells in gastric MALT lymphoma and its relationship between bcl-2 and p53 gene expression. Methods: TdT-mediated dUTP biotin Nick End labeling (TUNEL) and immuno-histochemistry ABC method were used to display apoptotic cells and the gene protein expression of bcl-2 and p53 independently. Results: Apoptotic indices (AI) in high-grade MALT lymphomas were significantly higher than in mixed-grade group and low-grade group (P<0.05). Bcl-2 was expressed in 83% of low-grade tumors, 61.6% of the median-grade tumors and 43.7% of high-grade tumors. An inverse correlation was observed between the expression of bcl-2 and apoptotic indices. Only 27 cases were p53 positive. The frequency of p53 positivity was significantly increased as the histologic grade advanced (P<0.05). There was also an inverse correlation between the expression of bcl-2 and p53. Conclusion: Apoptosis may be important in tumors development and transmission. P53 and bcl-2 were important regulatory genes of apoptosis and may be associated with transformation from low-grade to high-grade lymphomas.

  17. dNTP deficiency induced by HU via inhibiting ribonucleotide reductase affects neural tube development.

    Science.gov (United States)

    Guan, Zhen; Wang, Xiuwei; Dong, Yanting; Xu, Lin; Zhu, Zhiqiang; Wang, Jianhua; Zhang, Ting; Niu, Bo

    2015-02-03

    Exposure to environmental toxic chemicals in utero during the neural tube development period can cause developmental disorders. To evaluate the disruption of neural tube development programming, the murine neural tube defects (NTDs) model was induced by interrupting folate metabolism using methotrexate in our previous study. The present study aimed to examine the effects of dNTP deficiency induced by hydroxyurea (HU), a specific ribonucleotide reductase (RNR) inhibitor, during murine neural tube development. Pregnant C57BL/6J mice were intraperitoneally injected with various doses of HU on gestation day (GD) 7.5, and the embryos were checked on GD 11.5. RNR activity and deoxynucleoside triphosphate (dNTP) levels were measured in the optimal dose. Additionally, DNA damage was examined by comet analysis and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay. Cellular behaviors in NTDs embryos were evaluated with phosphorylation of histone H3 (PH-3) and caspase-3 using immunohistochemistry and western blot analysis. The results showed that NTDs were observed mostly with HU treatment at an optimal dose of 225 mg/kg b/w. RNR activity was inhibited and dNTP levels were decreased in HU-treated embryos with NTDs. Additionally, increased DNA damage, decreased proliferation, and increased caspase-3 were significant in NTDs embryos compared to the controls. Results indicated that HU induced murine NTDs model by disturbing dNTP metabolism and further led to the abnormal cell balance between proliferation and apoptosis.

  18. Effects of cinnamon (Cinnamomum zeylanicum) bark oil on testicular antioxidant values, apoptotic germ cell and sperm quality.

    Science.gov (United States)

    Yüce, A; Türk, G; Çeribaşi, S; Sönmez, M; Çiftçi, M; Güvenç, M

    2013-08-01

    Cinnamon and its contents have multifactorial properties such as antioxidant, anti-inflammatory and antidiabetic. Male infertility is one of the major health problems in life. The aim of this study was to investigate the effects of long-term cinnamon bark oil (CBO) ingestion on testicular antioxidant values, apoptotic germ cell and sperm quality of adult rats. Twelve male healthy Wistar rats were divided into two groups, each group containing six rats. While olive oil was given to control group, 100 mg kg(-1)  CBO was administered to the other group by gavage daily for 10 weeks. Body and reproductive organ weights, sperm characteristics, testicular lipid peroxidation and antioxidant enzyme activities, and testicular apoptosis via terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method were examined. A significant decrease in malondialdehyde level and marked increases in reduced glutathione level, glutathione peroxidase and catalase activities were observed in rats treated with CBO compared with the control group. CBO consumption provided a significant increase in weights of testes and epididymides, epididymal sperm concentration, sperm motility and diameter of seminiferous tubules when compared with the control group. However, CBO consumption tended to decrease the abnormal sperm rate and apoptotic germ cell count, but it did not reach statistical significance. It is concluded that CBO has improvement effect on testicular oxidant-antioxidant balance and sperm quality, and its consumption may be useful for asthenozoospermic men.

  19. Distinct Responses of Cytotoxic Ganoderma lucidum Triterpenoids in Human Carcinoma Cells.

    Science.gov (United States)

    Ruan, Weimei; Wei, Ying; Popovich, David G

    2015-11-01

    The medicinal mushroom Ganoderma lucidum is well recognized for its effective cancer-preventative and therapeutic properties, while specific components responsible for these anticancer effects are not well studied. Six triterpenoids that are ganolucidic acid E, lucidumol A, ganodermanontriol, 7-oxo-ganoderic acid Z, 15-hydroxy-ganoderic acid S, and ganoderic acid DM were isolated and identified from an extract of the mushroom. All compounds reduced cell growth in three human carcinoma cells (Caco-2, HepG2, and HeLa cells) dose dependently with LC50s from 20.87 to 84.36 μM. Moreover, the six compounds induced apoptosis in HeLa cells with a maximum increase (22%) of sub-G1 accumulations and 43.03% apoptotic cells in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (15-hydroxy-ganoderic acid S treatment). Apoptosis was further confirmed by annexin-V staining. Four of the compounds also caused apoptosis in Caco-2 cells with maximum 9.5% increase of sub-G1 accumulations (7-oxo-ganoderic acid Z treatment) and maximum 29.84% apoptotic cells in TUNEL assay (ganoderic acid DM treatment). Contrarily, none of the compounds induced apoptosis in HepG2 cells. The different responses of the three cell lines following these treatments indicated that the bioactive properties of these compounds may vary from cells of different sites of origin and are likely acting under diverse regulatory mechanisms.

  20. Time-course and molecular mechanism of hepatotoxicity induced by 1,3-dichloro-2-propanol in rats.

    Science.gov (United States)

    Lee, In-Chul; Ko, Je-Won; Lee, Sang-Min; Kim, Sung-Hwan; Shin, In-Sik; Moon, Og-Sung; Yoon, Won-Kee; Kim, Hyoung-Chin; Kim, Jong-Choon

    2015-07-01

    This study investigated the time-course of 1,3-dichloro-2-propanol (1,3-DCP)-induced hepatotoxicity and the molecular mechanism of its oxidative stress and apoptotic changes in rats. Thirty-six male rats were randomly assigned to six groups of six rats each and were administered a single oral dose of 1,3-DCP (90 mg/kg) or its vehicle. 1,3-DCP caused acute hepatic damage, as evidenced by marked increases in serum aminotransferase, alkaline phosphatase, and histopathological alterations. These functional and histopathological changes in the liver peaked at 12h after administration and then decreased progressively. Oxidative stress indices were increased significantly at 6h, peaked at 12h, and then decreased progressively. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)- and caspase-3-positive cells increased after 6h, peaked at 12 and 24h, and then decreased. The protein levels of phosphorylated mitogen-activated protein kinases (MAPKs) including p-Erk1/2 and p-JNK showed a similar trend to the numbers of TUNEL- and caspase-3-positive cells. These results indicate that 1,3-DCP increases oxidative stress, nuclear translocation of Nrf2, and expression of Nrf2-targeted genes, followed by increased functional and histopathological alterations in the liver. The increase in hepatocellular apoptosis induced by 1,3-DCP may be related to oxidative stress-mediated MAPK activation.

  1. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage

    Directory of Open Access Journals (Sweden)

    Carol Coughlan

    2015-01-01

    Full Text Available Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM. To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

  2. Comparative proteomic profiling and possible toxicological mechanism of acute injury induced by carbon ion radiation in pubertal mice testes

    Science.gov (United States)

    Zhang, Hong

    2016-07-01

    We investigated potential mechanisms of acute injury in pubertal mice testes after exposure to carbon ion radiation (CIR). Serum testosterone was measured following whole-body irradiation with a 2Gy carbon ion beam. Comparative proteomic profiling and Western blotting were applied to identify potential biomarkers and measure protein expression, and terminal dUTP nick end-labeling (TUNEL) was performed to detect apoptotic cells. Immunohistochemistry and immunofluorescence were used to investigate protein localization. Serum testosterone was lowest at 24h after CIR, and 10 differentially expressed proteins were identified at this time point that included eIF4E, an important regulator of initiation that combines with mTOR and 4EBP1 to control protein synthesis via the mTOR signalling pathway during proliferation and apoptosis. Protein expression and localization studies confirmed their association with acute injury following exposure to CIR. These three proteins may be useful molecular markers for detecting abnormal spermatogenesis following exposure to environmental and cosmic radiation

  3. The effects of erdosteine, N-acetylcysteine, and vitamin E on nicotine-induced apoptosis of hippocampal neural cells.

    Science.gov (United States)

    Demiralay, Rezan; Gürsan, Nesrin; Erdem, Havva

    2008-08-01

    This study investigated the frequency of apoptosis in rat hippocampal neural cells after intraperitoneal nicotine injection, examining the roles of the inflammatory markers myeloperoxidase (MPO) and tumor necrosis factor alpha (TNF-alpha) in nicotine-induced brain damage and the protective effects of three known antioxidant agents, N-acetylcysteine (NAC), erdosteine, and vitamin E. Female Wistar rats were divided into seven groups, each composed of nine rats: 2 negative control groups, 2 positive control groups, one erdosteine-treated group (500 mg/kg), one NAC-treated group (500 mg/kg), and one vitamin E-treated group (500 mg/kg). Nicotine was intraperitoneally injected at a dosage of 0.6 mg/kg for 21 days. Following nicotine injection, the antioxidants were administered orally; treatment was continued until the rats were killed. Apoptosis level in hippocampal neural cells was determined by using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling) method. Staining of cytoplasmic TNF-alpha in hippocampal neural cells and hippocampus MPO activity were evaluated by immunohistochemistry. Nicotine administration had no effect on local TNF-alpha production, or hippocampal MPO activity. The treatments with erdosteine, NAC and vitamin E significantly reduced the rate of nicotine-induced hippocampal neural cell apoptosis. This findings suggest that erdosteine and NAC can be as effective as vitamin E in protecting against nicotine-induced hippocampal neural cell apoptosis.

  4. Menoprogen, a TCM Herbal Formula for Menopause, Increases Endogenous E2 in an Aged Rat Model of Menopause by Reducing Ovarian Granulosa Cell Apoptosis

    Directory of Open Access Journals (Sweden)

    Yu Li

    2016-01-01

    Full Text Available The effect of Menoprogen (MPG on ovarian granulosa cell (GC apoptosis was investigated in vitro and in vivo in an aged rat model of menopause. Intragastric administration of Menoprogen or estradiol valerate to 14-month-old senile female rats for eight weeks increased plasma E2 levels, as well as the weight of both ovarian and uterine tissues. Flow cytometric (FCM analysis of isolated GCs from MPG-treated aged rats showed reductions in the G0/G1 ratio and apoptotic peaks. Isolated GCs also exhibited an increase in cell size and the number of cytoplastic organelles and intracellular gap junctions, the reappearance of secretory granules, and a lack of apoptotic bodies as determined by TEM. Results from a TdT-mediated dUTP nick end-labeling (TUNEL assay revealed a reduction in TUNEL-positive GCs after MPG treatment. Immunohistochemical analysis showed a downregulation of proapoptotic Bax proteins and an upregulation of antiapoptotic Bcl-2 proteins. The addition of MPG-medicated serum to the media of cultured GCs also reduced cadmium chloride-induced apoptosis and downregulated caspase-3 protein expression. This work demonstrates that Menoprogen inhibits GC apoptosis in aged female rats and thereby increases E2 production. This represents a novel mechanism of action for this herbal medicine in the treatment of menopausal symptoms.

  5. Study on Effect of IH764-3, an Active Principle of Salviae miltiorrhizae, in Inducing Hepatic Stellate Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    赵东强; 姜慧卿; 修贺明; 张晓岚

    2002-01-01

    Objective: To explore the anti-fibrotic mechanism of Salviae miltiorrhizae from the view of proliferation and apoptosis of hepatic stellate cells (HSC).Methods: IH764-3, an active principle of Salviae miltiorrhizae, was used to intervene in the cultured HSC in vitro. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method, and the cell apoptosis was examined by electron microscopy, flow cytometer and terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling method (TUNEL).Results: MTT showed that IH764-3 has obvious inhibition on the proliferation of HSC. Specific cell apoptosis figures of HSC, such as chromatin agglutination, were seen under electron microscopy in the IH764-3 treated group. By flow cytometer, it was shown that the HSC apoptosis rate in the IH764-3 treated group was higher than that in the control group, and the apoptosis inducing effect of IH764-3 was dose- and time-dependent. TUNEL analysis showed that the HSC apoptotsis rate was 28.3±1.5% after being incubated for 48 hrs with IH764-3, which was significantly higher than that in the control group (6.7±0.6%, P<0.05).Conclusion: IH764-3 could inhibit the proliferation of HSC and induce its apoptosis. These effects may be one of the anti-fibrotic mechanisms of Salviae miltiorrhizae.

  6. Antenatal taurine reduces cerebral cell apoptosis in fetal rats with intrauterine growth restriction*

    Institute of Scientific and Technical Information of China (English)

    Jing Liu; Xiaofeng Wang; Ying Liu; Na Yang; Jing Xu; Xiaotun Ren

    2013-01-01

    From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12th day of pregnancy, 300 mg/kg rine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neo-natal rats at 6 hours after delivery, neonatal rats with intrauterine growth restriction, and neonatal rats with intrauterine growth restriction undergoing taurine supplement were obtained for further experiments. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling assay revealed that the number of apoptotic cel s in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cel apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. nohistochemical staining revealed that taurine supplement increased glial cel line-derived neuro-trophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cel apoptosis through the glial cel line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.

  7. Pouterin, a novel potential cytotoxic lectin-like protein with apoptosis-inducing activity in tumorigenic mammalian cells.

    Science.gov (United States)

    Boleti, Ana Paula de A; Ventura, Cláudio A; Justo, Giselle Z; Silva, Rodrigo A; de Sousa, Ana Carolina T; Ferreira, Carmen V; Yano, Tomomasa; Macedo, Maria Lígia R

    2008-06-15

    In this study, the cytotoxicity of pouterin in tumorigenic and non-tumorigenic mammalian cell lines was investigated. We found that HeLa, Hep-2 and HT-29 tumor cells were highly sensitive to pouterin cytotoxicity in a dose-dependent manner, whereas non-tumorigenic Vero cells and human lymphocytes were relatively resistant to the protein. Among the tumor cell lines, HeLa cells showed the highest susceptibility to pouterin cytotoxicity, exhibiting a time-dependent increase in LDH leakage and an IC(50) value of 5mug/mL. Morphological alterations such as rounding, cell shrinkage and chromatin condensation, consistent with apoptotic cell death were observed. Apoptosis induction was demonstrated by DNA fragmentation as detected by terminal dUTP nick-end labeling (TUNEL). Furthermore, HeLa cells incubated with pouterin showed disruption of the actin cytoskeleton. Western blot analysis revealed that pouterin caused increased expression of p21, thus indicating cell cycle arrest. Subsequent studies provided evidence that apoptosis may be partially explained in the activation of the tumor necrosis factor receptor 1 (TNFR1) signaling. Interestingly, a time-dependent decrease of the expression of p65 nuclear factor kappa B (NFkappaB) subunit, concomitant with a downregulation of the inhibitor of apoptosis protein 1 (IAP1) was observed, suggesting that TNFR-mediated apoptosis is the predominant pathway induced by pouterin in HeLa cells.

  8. Cisplatin cytotoxicity of auditory cells requires secretions of proinflammatory cytokines via activation of ERK and NF-kappaB.

    Science.gov (United States)

    So, Hongseob; Kim, HyungJin; Lee, Jeong-Han; Park, Channy; Kim, Yunha; Kim, Eunsook; Kim, Jin-Kyung; Yun, Ki-Jung; Lee, Kang-Min; Lee, Haa-Yung; Moon, Sung-Kyun; Lim, David J; Park, Raekil

    2007-09-01

    The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, through the activation of ERK and NF-kappaB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-alpha. Immunohistochemical studies revealed that TNF-alpha expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-kappaB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-alpha, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

  9. Ethanolic extract of propolis induces apoptosis of HL-60 cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yan Shi; Yana Li; Co-first author Naie Li; Min Yu; Dong Wang; Lijun Kong

    2016-01-01

    Objective The aim of the study was to investigate whether ethanolic extract of propolis inhibits the growth and induces apoptosis of HL-60 cel s. Methods HL-60 cel s were treated for 24, 48, 72 h with various concentrations ethanolic extracts of prop-olis (0, 50, 100, and 200 μg/mL). The proliferation of HL-60 cels was determined using the 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, Hochest 33258 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to test the apoptosis of HL-60 cel s. We observed the expression levels of Bax and Bcl-2 in HL-60 cel s by immunohistochemistry. Results MTT assay showed that various concentrations of ethanolic extract of propolis had significant inhibitory efect on HL-60 cel proliferation (P Conclusion Ethanolic extract of propolis inhibits leukemia cel proliferation and induces apoptosis in vitro. Its mechanism may be related to the regulation of Bax and Bcl-2 expression and up-regulation of Bcl-2/Bax ratio.

  10. Environmental Enrichment Attenuated Sevoflurane-Induced Neurotoxicity through the PPAR-γ Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yupeng Zhao

    2015-01-01

    Full Text Available Sevoflurane is the most widely used inhaled anesthetic. Environmental enrichment (EE can reverse sevoflurane-induced learning and memory impairment in young mice. However, the mechanism by which EE elicits this effect is unclear. The peroxisome proliferator-activated receptor (PPAR regulatory pathway plays a critical role in the regulation of inflammation in central nervous system diseases. In this study, we investigated whether EE attenuates sevoflurane-induced learning and memory disability via the PPAR signaling pathway. Six-day-old mice were treated with 3% sevoflurane for 2 hours daily from postnatal day 6 (P6 to P8. Then, the mice were treated with EE. The effects of sevoflurane on learning and memory function, PPAR-γ expression in the brain, and the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and 5-bromodeoxyuridine-positive cells in the hippocampus were determined. Sevoflurane induced neuronal apoptosis and neurogenesis inhibition, which may impair learning and memory in young mice. Furthermore, sevoflurane downregulated PPAR-γ expression. Both EE and the PPAR-γ agonist, rosiglitazone, attenuated sevoflurane-induced neuronal apoptosis, neurogenesis inhibition, and learning and memory impairment. Our findings suggest that EE ameliorated sevoflurane-induced neurotoxicity and learning and memory impairment through the PPAR-γ signaling pathway. PPAR-γ may be a potential therapeutic target for preventing or treating sevoflurane-induced neurotoxicity.

  11. In vivo programmed cell death of Entamoeba histolytica trophozoites in a hamster model of amoebic liver abscess.

    Science.gov (United States)

    Villalba-Magdaleno, José D'Artagnan; Pérez-Ishiwara, Guillermo; Serrano-Luna, Jesús; Tsutsumi, Víctor; Shibayama, Mineko

    2011-05-01

    Entamoeba histolytica trophozoites can induce host cell apoptosis, which correlates with the virulence of the parasite. This phenomenon has been seen during the resolution of an inflammatory response and the survival of the parasites. Other studies have shown that E. histolytica trophozoites undergo programmed cell death (PCD) in vitro, but how this process occurs within the mammalian host cell remains unclear. Here, we studied the PCD of E. histolytica trophozoites as part of an in vivo event related to the inflammatory reaction and the host-parasite interaction. Morphological study of amoebic liver abscesses showed only a few E. histolytica trophozoites with peroxidase-positive nuclei identified by terminal deoxynucleotidyltransferase enzyme-mediated dUTP nick end labelling (TUNEL). To better understand PCD following the interaction between amoebae and inflammatory cells, we designed a novel in vivo model using a dialysis bag containing E. histolytica trophozoites, which was surgically placed inside the peritoneal cavity of a hamster and left to interact with the host's exudate components. Amoebae collected from bags were then examined by TUNEL assay, fluorescence-activated cell sorting (FACS) and transmission electron microscopy. Nuclear condensation and DNA fragmentation of E. histolytica trophozoites were observed after exposure to peritoneal exudates, which were mainly composed of neutrophils and macrophages. Our results suggest that production of nitric oxide by inflammatory cells could be involved in PCD of trophozoites. In this modified in vivo system, PCD appears to play a prominent role in the host-parasite interaction and parasite cell death.

  12. Salt Stress-induced Programmed Cell Death in Rice Root Tip Cells

    Institute of Scientific and Technical Information of China (English)

    Jian-You Li; Ai-Liang Jiang; Wei Zhang

    2007-01-01

    Salt stressed rice root tips were used to investigate the changes of reactive oxygen species (ROS) and antioxidant enzymes at the early stages of programmed cell death (PCD). The results indicated that 500 mmol/L NaCl treatment could lead to specific features of PCD in root tips, such as DNA ladder, nuclear condense and deformation, and transferase mediated dUTP nick end labeling positive reaction, which were initiated at 4 h of treatment and progressed thereafter. Cytochrome c release from mitochondria into cytoplasm was also observed, which occurred at 2 h and was earlier than the above nuclear events. In the very early phase of PCD, an immediate burst in hydrogen peroxide and superoxide anion production rate was accompanied by two-phase changes of superoxide dismutases and ascorbate peroxidase. A short period of increase in the activity was followed by prolonged impairment. Thus,we conclude that salt can induce PCD in rice root tip cells, and propose that in the early phase of rice root tip cell PCD, salt stress-induced oxidative burst increased the antioxidant enzyme activity, which, In turn, scavenged the ROS and abrogated PCD. Also, when the stress is prolonged, the antioxidant system is damaged and accumulated ROS induces the PCD process, which leads to cytochrome c release and nuclear change.

  13. Development of a novel nitro-derivative of noscapine for the potential treatment of drug-resistant ovarian cancer and T-cell lymphoma.

    Science.gov (United States)

    Aneja, Ritu; Vangapandu, Surya N; Lopus, Manu; Chandra, Ramesh; Panda, Dulal; Joshi, Harish C

    2006-06-01

    We have shown previously that an antitussive plant alkaloid, noscapine, binds tubulin, displays anticancer activity, and has a safe pharmacological profile in humans. Structure-function analyses pointed to a proton at position-9 of the isoquinoline ring that can be modified without compromising tubulin binding activity. Thus, many noscapine analogs with different functional moieties at position-9 were synthesized. Those analogs that kill human cancer cells resistant to other antimicrotubule agents, vincas and taxanes, were screened. Here, we present one such analog, 9-nitro-noscapine (9-nitro-nos), which binds tubulin and induces apoptosis selectively in tumor cells (ovarian and T-cell lymphoma) resistant to paclitaxel, vinblastine, and teniposide. 9-Nitro-nos treatment at doses as high as 100 microM did not affect the cell cycle profile of normal human fibroblasts. This selectivity of 9-nitro-nos for cancer cells represents a unique edge over the other available antimitotics. 9-Nitro-nos perturbs the progression of cell cycle by mitotic arrest, followed by apoptotic cell death associated with increased caspase-3 activation and appearance of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. Thus, we conclude that 9-nitro-nos has great potential to be a novel therapeutic agent for ovarian and T-cell lymphoma cancers, even those that have become drug-resistant to currently available chemotherapeutic drugs.

  14. Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine

    Directory of Open Access Journals (Sweden)

    Mohammad Rasoul Khazaei

    2016-09-01

    Full Text Available Objective(s: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. Materials and Methods:In this in vitro study, endometrial biopsies from endometriosis patients (n=9 were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml. Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0, 10, 25, 50 and 100 micromole/liter (µM concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO–Ethidium Bromide (EB double staining and Terminal deoxynucleotidyl transferase (TdT dUTP Nick-End Labeling (TUNEL assay. Data were analyzed by one-way ANOVA. Results: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P

  15. Salidroside attenuates myocardial ischemia-reperfusion injury via PI3K/Akt signaling pathway.

    Science.gov (United States)

    Xu, Mao-Chun; Shi, Hai-Ming; Gao, Xiu-Fang; Wang, Hao

    2013-01-01

    To investigate the cardioprotective effects of salidroside on myocardial ischemia-reperfusion injury (IRI) in rabbits and the underlying action mechanisms in PI3K/Akt signaling pathway, a rabbit ischemia/reperfusion model was created by ligating the left anterior descending coronary arterial branch for 30 min and by releasing the ligature to allow reperfusion for 120 min. Salidroside or salidroside+PI3K inhibitor (LY294002) was administered via intracoronary injections at the onset of reperfusion. Apoptosis of cardiomyocytes was assessed by terminal dUTP nick-end labeling assay, and the expression of apoptosis-related proteins was observed by immunohistochemistry. The expressions of total Akt and phosphorylated Akt (p-Akt) were detected by western blot analysis. The results showed that intracoronary injection of salidroside at the onset of reperfusion markedly reduced the apoptosis of cardiomyocytes, significantly increasing Bcl-2 and p-Akt proteins expressions and decreasing Bax and caspase-3 expressions in the hearts subjected to ischemia followed by 120-min reperfusion. However, the anti-apoptotic effect induced by salidroside was inhibited by LY294002, which blocked the activation of Akt. These results suggested that intracoronary administration of salidroside at the onset of reperfusion could significantly reduce the IRI-induced apoptosis of cardiomyocytes, and this protective mechanism seemed to be mediated by the PI3K-Akt signaling pathway.

  16. The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

    Institute of Scientific and Technical Information of China (English)

    Fei GAO; Jing YI; Jing Qi YUAN; Gui Ying SHI; Xue Ming TANG

    2004-01-01

    Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate,to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry,with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide (PI),indicated that As2O3 (2 μM) could induce apoptosis in NB4 cells prevailingly from G2/M phase,and this efficacy was enhanced upon co-administration of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) (2.5 μM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells' susceptibility to apoptosis by As2O3.

  17. Hydrogen-rich saline reduces cell death through inhibition of DNA oxidative stress and overactivation of poly (ADP-ribose) polymerase-1 in retinal ischemia-reperfusion injury.

    Science.gov (United States)

    Liu, Hongwei; Hua, Ning; Xie, Keliang; Zhao, Tingting; Yu, Yonghao

    2015-08-01

    Overactivation of poly (ADP-ribose) polymerase 1 (PARP-1), as a result of sustained DNA oxidation in ischemia-reperfusion injury, triggers programmed cell necrosis and apoptosis. The present study was conducted to demonstrate whether hydrogen-rich saline (HRS) has a neuroprotective effect on retinal ischemia reperfusion (RIR) injury through inhibition of PARP-1 activation. RIR was induced by transient elevation of intraocular pressure in rats. HRS (5 ml/kg) was administered peritoneally every day from the beginning of reperfusion in RIR rats until the rats were sacrificed. Retinal damage and cell death was determined using hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. DNA oxidative stress was evaluated by immunofluorescence staining of 8-hydroxy-2-deoxyguanosine. In addition, the expression of PARP-1 and caspase-3 was investigated by western blot analysis and/or immunohistochemical staining. The results demonstrated that HRS administration improved morphological alterations and reduced apoptosis following RIR injury. Furthermore, the present study found that HRS alleviated DNA oxidation and PARP-1 overactivation in RIR rats. HRS can protect RIR injury by inhibition of PARP-1, which may be involved in DNA oxidative stress and caspase-3-mediated apoptosis.

  18. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer’s disease

    Institute of Scientific and Technical Information of China (English)

    Yan Wang; Biao Cai; Jing Shao; Ting-ting Wang; Run-ze Cai; Chang-ju Ma; Tao Han; Jun Du

    2016-01-01

    Genistein is effective against amyloid-βtoxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer’s disease. A rat model of Alzheimer’s disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-βpeptide (25–35). In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg) was given prior to establishing Alzheimer’s dis-ease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genist-ein in Alzheimer’s disease rats. These ifndings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

  19. Morphological and intracellular alterations induced by Serratia marcescens cytotoxin.

    Science.gov (United States)

    Carbonell, Gleize Villela; Falcón, Rosabel; Yamada, Aureo T; da Fonseca, Benedito Antonio Lopes; Yano, Tomomasa

    2004-01-01

    In the present work, in vitro assays were used to investigate the toxicity of Serratia marcescens cytotoxin in cultured Chinese hamster ovary (CHO) cells. The time necessary to detect cellular alterations such as the onset of apoptosis, the perturbation of mitochondrial function, and cytoskeletal changes was assessed. The internalization of the cytotoxin by CHO cells was also examined. Within 10-15 min of exposure to cytotoxin, CHO cells became round, the nucleus shrank, the chromatin became more compact, and cytoplasmic blebs appeared on the cell surface. TUNEL (TdT-mediated dUTP nick end labeling) and propidium iodide staining identified some nuclei with fragmented DNA, and electrophoresis of CHO cell DNA obtained after 30-min exposure to S. marcescens toxin showed a pattern of DNA fragments typically associated with apoptosis. The cells also lost their characteristic actin organization within 10 min of exposure to cytotoxin. Lactate dehydrogenase leakage was detected after 20-min exposure to the cytotoxin and increased with time thereafter. Concomitantly, there was a time-dependent reduction in mitochondrial activity. Fluorescein-labeled S. marcescens cytotoxin was detected only on the surface of CHO cells, even after 30-min exposure to the toxin. These results show that there was no internalization of the toxin by CHO cells, and that, once bound to the cell surface, the toxin was able to induce changes in intracellular metabolism and to trigger cell death by apoptosis.

  20. Clinicopathological significance and relations of Caspase-3 expression, cell proliferation and apoptosis in gastric cancer and the precancerous lesions%乳腺癌钼靶X线表现特征(118例分析)

    Institute of Scientific and Technical Information of China (English)

    XiaoBin Hu; Jing Zhao; Lin Yang; Yan Xin

    2009-01-01

    Objective: we investigated the relationship between the expression of Caspase-3. cell proiferation and apoptosis in gastric cancer and their precancerous lesions, to explore the tumorigenesis of the stomach mucosa. Methods: Caspase-3 expression in 13 normal gastric mucosa, 6 chronic atrophic gastritis (CAG), 31 intestinal metaplasia (IM), 114 dysplasia (DYS) and 20 gastric carcinomas were investigated immunohistochemically. Cell proliferation was evaluated with anti-Ki-67immunostaining and apoptosis was evaluated using DNA fragmentation in situ by TdT-mediated dUTP biotin nick end label-ing (TUNEL) method. Results: Caspase-3 mild-moderately positive expression was observed in most of normal superficial epithelia, its positively polar distribution in normal mucosa, CAG, IM, DYS and gastric carcinomas changed as seen in TU-NEL, and so did the positive rate. Caspase-3 protein expression showed significantly positive correlation with the number of apoptotic cells labeled with TUNEL (correlation coefficient r=0.94; P 0.05). Conclusion: Caspase-3 protein expression was up-regulated from CAG to IM and mild-moderate atypical dysplasia, but down-regulated in severe dysplasia and gastric carcinoma, indicating that inactivity or reduced expression of Caspase-3 is closely correlated with carcinogenesis of the stomach mucosa.

  1. N-methyl-D-aspartate receptor subtype 3A promotes apoptosis in developing mouse brain exposed to hyperoxia

    Institute of Scientific and Technical Information of China (English)

    Jimei Li; Shanping Yu; Zhongyang Lu; Osama Mohamad; Ling Wei

    2012-01-01

    In the present study, 7 day postnatal C57/BL6 wild-type mice (hyperoxia group) and 7 day postnatal N-methyl-D-aspartate receptor subtype 3A knockout mice (NR3A KO group) were exposed to 75% oxygen and 15% nitrogen in a closed container for 5 days. Wild-type mice raised in normoxia served as controls. TdT-mediated dUTP nick end labeling (TUNEL)/neuron-specific nuclear protein (NeuN) and 5-bromo-2'-deoxyuridine (BrdU)/NeuN immunofluorescence staining showed that the number of apoptotic cells and the number of proliferative cells in the dentate subgranular zone significantly increased in the hyperoxia group compared with the control group. However, in the same hyperoxia environment, the number of apoptotic cells and the number of proliferative cells significantly decreased in the NR3A KO group compared with hyperoxia group. TUNEL+/NeuN+ and BrdU+/NeuN+ cells were observed in the NR3A KO and the hyperoxia groups. These results demonstrated that the NR3A gene can promote cell apoptosis and mediate the potential damage in the developing brain induced by exposure to non-physiologically high concentrations of oxygen.

  2. Epigallocatechin-3-gallate suppresses the lipid deposition through the apoptosis during differentiation in bovine bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Jeong, Jin Young; Suresh, Sekar; Jang, Mi; Park, Mi Na; Gobianand, Kuppannan; You, Seungkwon; Yeon, Sung-Heom; Lee, Hyun-Jeong

    2015-01-01

    Epigallocatechin gallate (EGCG), a major component of tea, has known effects on obesity, fatty liver, and obesity-related cancer. We explored the effects of EGCG on the differentiation of bovine mesenchymal stem cells (BMSCs, which are multipotent) in a dose- and time-dependent manner. Differentiating BMSCs were exposed to various concentrations of EGCG (0, 10, 50, 100, and 200 µM) for 2, 4, and 6 days. BMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/high-glucose medium with adipogenic inducers for 6 days, and the expression levels of various genes involved in adipogenesis were measured using real-time polymerase chain reaction (PCR) and Western blotting. We assessed apoptosis by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining of control and EGCG-exposed cells. We found that EGCG significantly suppressed fat deposition and cell viability (P EGCG during adipogenic differentiation (P EGCG lowered the expression levels of the adipogenic proteins encoded by these genes (P EGCG induced apoptosis during adipogenic differentiation (P EGCG potentially inhibits adipogenesis by triggering apoptosis; the data suggest that EGCG inhibits adipogenic differentiation in BMSCs. © 2014 The Authors. Published by John Wiley & Sons Ltd on behalf of the International Federation for Cell Biology.

  3. Brief left ventricular pressure overload reduces myocardial apoptosis.

    Science.gov (United States)

    Huang, Hsien-Hao; Lai, Chang-Chi; Chiang, Shu-Chiung; Chang, Shi-Chuan; Chang, Chung-Ho; Lin, Jin-Ching; Huang, Cheng-Hsiung

    2015-03-01

    Both apoptosis and necrosis contribute to cell death after myocardial ischemia and reperfusion. We previously reported that brief left ventricular pressure overload (LVPO) decreased myocardial infarct (MI) size. In this study, we investigated whether brief pressure overload reduces apoptosis and the mechanisms involved. MI was induced by a 40-min occlusion of the left anterior descending coronary artery and 3-h reperfusion in male anesthetized Sprague-Dawley rats. Brief LVPO was achieved by two 10-min partial snarings of the ascending aorta, raising the systolic left ventricular pressure 50% above the baseline value. Ischemic preconditioning was elicited by two 10-min coronary artery occlusions and 10-min reperfusions. Brief LVPO and ischemic preconditioning significantly decreased MI size (P Brief pressure overload significantly reduced myocardial apoptosis, as evidenced by the decrease in the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei (P brief pressure overload significantly increased Bcl-2 (P brief pressure overload (P Brief left LVPO significantly reduces myocardial apoptosis. The underlying mechanisms might be related to modulation of Bcl-2 and Bax, inhibition of p53, increased Akt phosphorylation, and suppressed c-Jun N-terminal kinase phosphorylation. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Fusarenon-X-induced apoptosis in the liver, kidney, and spleen of mice

    Science.gov (United States)

    Sutjarit, Samak; Poapolathep, Amnart

    2016-01-01

    Fusarenon-X is a non-macrocyclic type B trichothecene mycotoxin. It occurs naturally in agricultural commodities, such as wheat and barley. We investigated fusarenon-X-induced apoptosis in the liver, kidney, and spleen of male and female mice after a single exposure. Thus, mice were orally administered fusarenon-X (4 mg/kg body weight) and were assessed at 0, 3, 9, 18, 24, and 48 hours after treatment. Apoptosis in the liver, kidney, and spleen was determined using hematoxylin and eosin staining, the terminal deoxynucleotidyl transferase dUTP nick end labeling method, immunohistochemistry for proliferating cell nuclear antigen, and electron microscopy. Fusarenon-X-induced apoptosis at 9 hours after treatment, particularly hepatocytes around the central lobular zone of the liver, in proximal tubular cells of the kidney, and in hematopoietic cells in the red pulp area of the spleen in both male and female mice. The results of this study should be very useful with regard to the toxicity of fusarenon-X in both humans and domestic animals, which has been attributed to the intake of food contaminated with mycotoxins, especially fusarenon-X. PMID:27559248

  5. The apoptotic effect of a high dose of toluene on liver tissue during the acute phase: an experimental study.

    Science.gov (United States)

    Ayan, Murat; Tas, Ufuk; Sogut, Erkan; Kuloglu, Tuncay; Cayli, Sevil; Kocaman, Nevin; Karaca, Zafer Ismail; Sahin, Mehmet

    2013-09-01

    The aim of this study is to investigate the acute toxic effects of high-dose toluene and its mechanisms on the liver tissue of toluene-treated rats. In this study, 16 adult male Wistar albino rats (200-220 g) were divided into two equal groups. Group I was used as a control group, while group II was exposed to high dose of toluene, 5200 mg/kg (6 ml/kg per gavage). After the 3-hour experimental period, blood samples and liver tissues were taken from the euthanized animals. Serum aspartate and alanine aminotransferase levels were assayed. Liver tissues were fixed in 10% neutral formalin, then embedded in paraffin and sectioned (5 μm thickness). Sections were stained with hematoxylin and eosin for histopathological examination. A terminal transferase dUTP nick end labeling assay was also done for the determination of apoptosis in liver tissues. For the determination of Bax and caspase-3 immunoreactivity, the sections were stained using avidin-biotin-peroxidase immunohistochemical method. The level of plasma transaminase was found to be increased in toluene administered rats. Additionally, slight degeneration of hepatocyte and mononuclear cell infiltration was observed in the liver tissue sections and a high (+++) immunoreactivity for Bax and caspase-3 protein was observed in the toluene group. This study showed that the high dose of toluene triggers apoptosis in the liver of rats via the mitochondrial pathway in acute period.

  6. Fenugreek, a naturally occurring edible spice, kills MCF-7 human breast cancer cells via an apoptotic pathway.

    Science.gov (United States)

    Khoja, Kholoud K; Shaf, Gowhar; Hasan, Tarique N; Syed, Naveed Ahmed; Al-Khalifa, Abdrohman S; Al-Assaf, Abdullah H; Alshatwi, Ali A

    2011-01-01

    There is growing use of anticancer complementary and alternative medicines worldwide. Trigonella foenum graecum (Fenugreek) is traditionally applied to treat disorders such as diabetes, high cholesterol, wounds, inflammation, and gastrointestinal ailments. Fenugreek is also reported to have anticancer properties due to its active beneficial chemical constituents. The mechanism of action of several anticancer drugs is based on their ability to induce apoptosis. The objective of the study was to characterize the downstream apoptotic genes targeted by FCE in MCF-7 human immortalized breast cells. FCE effectively killed MCF-7 cells through induction of apoptosis,confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and RT-PCR assays. When cells were exposed to 50 μg/mL FCE for 24 hours, 23.2% apoptotic cells resulted, while a 48-hour exposure to 50 μg/mL caused 73.8% apoptosis. This was associated with increased expression of Caspase 3, 8, 9, p53, Fas, FADD, Bax and Bak in a time-and dose-dependent manner, as determined by real- time quantitative PCR. In summary, the induction of apoptosis by FCE is effected by its ability to increase the expression of pro-apoptotic genes and the spice holds promise for consideration in complementary therapy for breast cancer patients.

  7. Experimental Study on the Expression of HIF-1α and Its Relationship to Apoptosis in Tissues around Cerebral Bleeding Loci

    Institute of Scientific and Technical Information of China (English)

    朱遂强; 唐洲平; 郭守刚; 彭岚; 方思羽; 张苏明

    2004-01-01

    The expression of hypoxia inducible factor-1 alpha (HIF-1α) and its relationship to apoptosis in tissues around cerebral bleeding loci was studied. The expression of HIF-1α and apoptosis in 37 samples of tissues around cerebral bleeding loci and 9 samples of normal cerebral tissues was assessed by immunohistochemical straining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling methods. In 37 tissue samples around cerebral bleeding loci, the positive rate of the HIF-1α expression was 40.6 %. Especially in the patients with amount of bleeding>60 ml, the positive rate (88.9 %) of the HIF-1α expression was significantly higher than those with the amount of bleeding ranging from 30-45 ml or 45-60 ml (P<0.05). The expression of HIF-1α was increased as the amount of bleeding and operative time increased (P<0.05). There existed a positive correlation between HIF-1α labeling index and apoptosis index (n= 12, r= 0.56, P<0.01). These results suggested that the expression of HIF-1α was closely related with the time of hemorrhage and the amount of bleeding, and could induce the apoptosis of neurons.

  8. Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.)

    Science.gov (United States)

    Wang, Shuping; Zhang, Gaisheng; Zhang, Yingxin; Song, Qilu; Chen, Zheng; Wang, Junsheng; Guo, Jialin; Niu, Na; Wang, Junwei; Ma, Shoucai

    2015-01-01

    Plant male sterility has often been associated with mitochondrial dysfunction; however, the mechanism in wheat (Triticum aestivum L.) has not been elucidated. This study set out to probe the mechanism of physiological male sterility (PHYMS) induced by the chemical hybridizing agent (CHA)-SQ-1, and cytoplasmic male sterility (CMS) of wheat at the proteomic level. A total of 71 differentially expressed mitochondrial proteins were found to be involved in pollen abortion and further identified by MALDI-TOF/TOF MS (matrix-assisted laser desorption/ionization-time of fight/time of flight mass spectrometry). These proteins were implicated in different cellular responses and metabolic processes, with obvious functional tendencies toward the tricarboxylic acid cycle, the mitochondrial electron transport chain, protein synthesis and degradation, oxidation stress, the cell division cycle, and epigenetics. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways involved in anther abortion and pollen defects. Accordingly, a mitochondria-mediated male sterility protein network in wheat is proposed; this network was further confirmed by physiological data, RT-PCR (real-time PCR), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. The results provide intriguing insights into the metabolic pathway of anther abortion induced by CHA-SQ-1 and also give useful clues to identify the crucial proteins of PHYMS and CMS in wheat. PMID:26136264

  9. Abnormal development of tapetum and microspores induced by chemical hybridization agent SQ-1 in wheat.

    Science.gov (United States)

    Wang, Shuping; Zhang, Gaisheng; Song, Qilu; Zhang, Yingxin; Li, Zheng; Guo, Jialin; Niu, Na; Ma, Shoucai; Wang, Junwei

    2015-01-01

    Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.

  10. Premature tapetum degeneration: a major cause of abortive pollen development in photoperiod sensitive genic male sterility in rice.

    Science.gov (United States)

    Shi, Yinlian; Zhao, Sha; Yao, Jialing

    2009-08-01

    Photoperiod-sensitive genic male-sterile (PSGMS) rice (Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT-mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.

  11. Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus x Graomys centralis male hybrids.

    Science.gov (United States)

    Rodriguez, Valeria; Diaz de Barboza, Gabriela; Ponce, Ruben; Merico, Valeria; Garagna, Silvia; Tolosa de Talamoni, Nori

    2010-01-01

    Spermatogenic impairment and the apoptotic pathways involved in establishing sterility of male hybrids obtained from crossing Graomys griseoflavus females with Graomys centralis males were studied. Testes from G. centralis, G. griseoflavus and hybrids were compared at different ages. Terminal transferase-mediated dUTP nick-end labelling assay (TUNEL), Fas, Bax and cytochrome c labelling were used for apoptosis evaluation, and calbindin D(28k) staining as an anti-apoptotic molecule. In 1-month-old animals, spermatocytes were positive for all apoptotic markers, but moderate TUNEL (+) spermatocyte frequency was only found in G. centralis. At subsequent ages, the apoptotic markers were downregulated in testes from parental cytotypes, but not in hybrid testes. TUNEL (+) spermatocytes were present at 78% and 44% per tubule cross-section in 2- and 3-month-old hybrid animals, respectively. Pachytene spermatocyte death in adult hybrids occurs via apoptosis, as revealed by high caspase-3 expression. Calbindin was highly expressed in spermatocytes of adult hybrids, in which massive cell death occurs via apoptosis. Calbindin co-localisation with TUNEL or Fas, Bax and cytochrome c was very limited, suggesting an inverse regulation of calbindin and apoptotic markers. Hybrid sterility is due to breakdown of spermatogenesis at the pachytene spermatocyte stage. Both extrinsic and intrinsic pathways are involved in apoptosis of spermatocytes, which are the most sensitive cell type to apoptotic stimuli.

  12. Evaluation of sperm protamine deficiency and apoptosis in infertile men with idiopathic teratozoospermia.

    Science.gov (United States)

    Dehghanpour, Fatemeh; Tabibnejad, Nasim; Fesahat, Farzaneh; Yazdinejad, Fatemeh; Talebi, Ali Reza

    2017-06-01

    Sperm morphology plays an important role in infertility, especially in cases of defects in the heads of spermatozoa. Tapered-head or elongated-head spermatozoa are examples of morphological abnormalities. The aim of this study was to compare the semen parameters, levels of protamine deficiency, and frequency of apoptosis between patients with normozoospermia and those with teratozoospermia with tapered-head spermatozoa. Fifty-two semen samples (27 patients with tapered-head sperm and 25 fertile men) were collected and semen analysis was performed according to the World Health Organization criteria for each sample. Protamine deficiency and the percentage of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3) staining and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays, respectively. Sperm concentration, motility, and normal morphology in the tapered-head spermatozoa (cases) were significantly lower than in the normozoospermic samples (controls). CMA3-reactive spermatozoa (CMA3+) in the case group were more common than in the controls. Apoptotic spermatozoa (TUNEL-positive) were significantly more common in the cases than in the controls. This analysis showed that tapered-head spermatozoa contained abnormal chromatin packaging and exhibited a high rate of apoptosis, which can be considered to be an important reason for the impaired fertility potential in teratozoospermic patients with tapered-head spermatozoa.

  13. Combinatorial MicroRNAs Suppress Hypoxia-Induced Cardiomyocytes Apoptosis

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    Yingqi Xu

    2015-09-01

    Full Text Available Background/Aims: Our previous in silico analysis revealed potential synergy in the activities of micro(miRNAs in myocardial infarction. The present study investigated whether miR-1 and -21 act synergistically to protect against cardiomyocytes apoptosis. Methods: Cell survival was analyzed with cell viability assay; apoptosis was detected by flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling, and the caspase-3 activity assay; and protein expression level was determined by western blotting. Results: MiR-1:miR-21 and several other miRNA pairs were evaluated for their potentially synergistic effects against myocardial hypoxia in neonatal rat ventricular cardiomyocytes. Lower combination indices suggested that miRNA pairs acted synergistically to inhibit apoptosis; miR-1 and -21 jointly blocked hypoxia-induced cardiomyocytes apoptosis. Moreover, combined application of miR-1 and -21 activated Akt and blocked hypoxia-induced upregulation of p53 in these cells. Conclusion: MiR-1 and -21 exert synergistic effects against hypoxia-induced cardiomyocytes apoptosis. These results provide a basis for the development of combined miRNA-based therapeutics to treat cardiovascular diseases.

  14. Treatment of steroid-induced osteonecrosis of the femoral head using porous Se@SiO2 nanocomposites to suppress reactive oxygen species

    Science.gov (United States)

    Deng, Guoying; Niu, Kerun; Zhou, Feng; Li, Buxiao; Kang, Yingjie; Liu, Xijian; Hu, Junqing; Li, Bo; Wang, Qiugen; Yi, Chengqing; Wang, Qian

    2017-01-01

    Reducing oxidative stress (ROS) have been demonstrated effective for steroid-induced osteonecrosis of the femoral head (steroid-induced ONFH). Selenium (Se) plays an important role in suppressing oxidative stress and has huge potential in ONFH treatments. However the Se has a narrow margin between beneficial and toxic effects which make it hard for therapy use in vivo. In order to make the deficiency up, a control release of Se (Se@SiO2) were realized by nanotechnology modification. Porous Se@SiO2 nanocomposites have favorable biocompatibility and can reduced the ROS damage effectively. In vitro, the cck-8 analysis, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) stain and flow cytometry analysis showed rare negative influence by porous Se@SiO2 nanocomposites but significantly protective effect against H2O2 by reducing ROS level (detected by DCFH-DA). In vivo, the biosafety of porous Se@SiO2 nanocomposites were confirmed by the serum biochemistry, the ROS level in serum were significantly reduced and the curative effect were confirmed by Micro CT scan, serum Elisa assay (inflammatory factors), Western blotting (quantitative measurement of ONFH) and HE staining. It is expected that the porous Se@SiO2 nanocomposites may prevent steroid-induced ONFH by reducing oxidative stress. PMID:28256626

  15. Dealcoholized Korean Rice Wine (Makgeolli) Exerts Potent Anti-Tumor Effect in AGS Human Gastric Adenocarcinoma Cells and Tumor Xenograft Mice.

    Science.gov (United States)

    Shin, Eun Ju; Kim, Sung Hee; Kim, Jae Ho; Ha, Jaeho; Hwang, Jin-Taek

    2015-09-01

    Makgeolli is a traditional wine in Korea and has been traditionally believed to exhibit health benefits. However, the inhibitory effect of dealcoholized makgeolli (MK) on cancer has never been investigated scientifically. In this study, MK exhibited an anti-angiogenic effect by inhibiting tube formation in human umbilical vein endothelial cells, without cytotoxicity. Treatment with MK reduced the proliferation of AGS human gastric adenocarcinoma cells in a dose-dependent manner and increased the sub-G1 population. Next, we evaluated whether MK could induce apoptosis in AGS cells by using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay or Annexin V method. Treatment with MK at 500 and 1,000 μg/ml increased the number of TUNEL-positive AGS cells. Under the same conditions, MK-treated (500 and 1,000 μg/ml) cells showed significant induction of early or late apoptosis, compared with untreated cells (no induction). In addition, MK also induced phosphatase and tensin homolog (PTEN) expression in AGS cells. However, p53 expression in AGS cells was not changed by MK treatment. Furthermore, MK at 500 mg/kg·d reduced the tumor size and volume in AGS tumor xenografts. Taken together, MK may be useful for the prevention of cancer cell growth.

  16. Apoptosis in Granulosa cells during follicular atresia:relationship with steroids and insulin-like growth factors

    Institute of Scientific and Technical Information of China (English)

    Yuan Song YU; Hong Shu SUI; Zheng Bin HAN; Wei LI; Ming Jiu LUO; Jing He TAN

    2004-01-01

    It is well known that during mammalian ovarian follicular development, the majority of follicles undergo atresia at various stages of their development. However, the mechanisms controlling this selection process remain unknown. In this study, we investigated apoptosis in granulosa cells during goat follicular atresia by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The changes in the levels of steroids, insulin-like growth factors (IGFs) and IGF receptors were studied by radioimmunoassay (RIA) and semi-quantitative reverse transcription-PCR. We found that the percentage of apoptotic granulosa cells in the atretic (A) follicles was significantly higher than that in the slightly atretic (SA) and healthy (H) follicles. The level of estradiol and the ratio of estradiol to progesterone in H follicles were significantly higher than those in A follicles. On the other hand, the level of progesterone was not significantly different among these follicle types. We also found that the level of IGF-I in H follicles was higher than in SA and A follicles, whereas the amount of IGF-Ⅱ did not vary significantly. The expression of IGF receptor also decreased in A follicles as compared to that in H and SA follicles. These results suggested that estradiol and IGF-I might be involved in controlling apoptosis in granulosa cells during follicular atresia.

  17. White matter apoptosis is increased by delayed hypothermia and rewarming in a neonatal piglet model of hypoxic ischemic encephalopathy.

    Science.gov (United States)

    Wang, B; Armstrong, J S; Reyes, M; Kulikowicz, E; Lee, J-H; Spicer, D; Bhalala, U; Yang, Z-J; Koehler, R C; Martin, L J; Lee, J K

    2016-03-01

    Therapeutic hypothermia is widely used to treat neonatal hypoxic ischemic (HI) brain injuries. However, potentially deleterious effects of delaying the induction of hypothermia and of rewarming on white matter injury remain unclear. We used a piglet model of HI to assess the effects of delayed hypothermia and rewarming on white matter apoptosis. Piglets underwent HI injury or sham surgery followed by normothermic or hypothermic recovery at 2h. Hypothermic groups were divided into those with no rewarming, slow rewarming at 0.5°C/h, or rapid rewarming at 4°C/h. Apoptotic cells in the subcortical white matter of the motor gyrus, corpus callosum, lateral olfactory tract, and internal capsule at 29h were identified morphologically and counted by hematoxylin & eosin staining. Cell death was verified by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay. White matter neurons were also counted, and apoptotic cells were immunophenotyped with the oligodendrocyte marker 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase). Hypothermia, slow rewarming, and rapid rewarming increased apoptosis in the subcortical white matter relative to normothermia (ppiglets had more apoptosis in the lateral olfactory tract than those that were rewarmed (ppiglets had more apoptosis than shams after normothermia, slow rewarming, and rapid rewarming (ppiglet model of HI; in some regions these temperature effects are independent of HI. Vulnerable cells include myelinating oligodendrocytes. This study identifies a deleterious effect of therapeutic hypothermia in the developing brain.

  18. Mast cells and hypoxia drive tissue metaplasia and heterotopic ossification in idiopathic arthrofibrosis after total knee arthroplasty.

    Science.gov (United States)

    Freeman, Theresa A; Parvizi, Javad; Dela Valle, Craig J; Steinbeck, Marla J

    2010-09-01

    Idiopathic arthrofibrosis occurs in 3-4% of patients who undergo total knee arthroplasty (TKA). However, little is known about the cellular or molecular changes involved in the onset or progression of this condition. To classify the histomorphologic changes and evaluate potential contributing factors, periarticular tissues from the knees of patients with arthrofibrosis were analyzed for fibroblast and mast cell proliferation, heterotopic ossification, cellular apoptosis, hypoxia and oxidative stress. The arthrofibrotic tissue was composed of dense fibroblastic regions, with limited vascularity along the outer edges. Within the fibrotic regions, elevated numbers of chymase/fibroblast growth factor (FGF)-expressing mast cells were observed. In addition, this region contained fibrocartilage and associated heterotopic ossification, which quantitatively correlated with decreased range of motion (stiffness). Fibrotic, fibrocartilage and ossified regions contained few terminal dUTP nick end labeling (TUNEL)-positive or apoptotic cells, despite positive immunostaining for lactate dehydrogenase (LDH)5, a marker of hypoxia, and nitrotyrosine, a marker for protein nitrosylation. LDH5 and nitrotyrosine were found in the same tissue areas, indicating that hypoxic areas within the tissue were associated with increased production of reactive oxygen and nitrogen species. Taken together, we suggest that hypoxia-associated oxidative stress initiates mast cell proliferation and FGF secretion, spurring fibroblast proliferation and tissue fibrosis. Fibroblasts within this hypoxic environment undergo metaplastic transformation to fibrocartilage, followed by heterotopic ossification, resulting in increased joint stiffness. Thus, hypoxia and associated oxidative stress are potential therapeutic targets for fibrosis and metaplastic progression of idiopathic arthrofibrosis after TKA.

  19. Alterations in the expression of atrial calpains in electrical and structural remodeling during aging and atrial fibrillation.

    Science.gov (United States)

    Xu, Guo-Jun; Gan, Tian-Yi; Tang, Bao-Peng; Chen, Zu-Heng; Mahemuti, Ailiman; Jiang, Tao; Song, Jian-Guo; Guo, Xia; Li, Yao-Dong; Zhou, Xian-Hui; Zhang, Yu; Li, Jin-Xin

    2013-11-01

    The aim of this study was to investigate the correlation between the change in the expression of atrial calpains and electrical, molecular and structural remodeling during aging and atrial fibrillation (AF). Adult and aged canines in sinus rhythm (SR) and with persistent AF (induced by rapid atrial pacing) were investigated. A whole-cell patch clamp was used to measure the L-type Ca2+ current (ICa-L) in cells in the left atrium. The mRNA and protein expression of the L-type calcium channel alc subunit (LVDCCa1c) and calpains were measured by quantitative (q)PCR and western blot analysis. Histopathological and ultrastructural changes were analyzed via light and electron microscopy. The quantity of apoptotic myocytes was determined by a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay. In SR groups, atrial cells of the aged canines exhibited a longer action potential (AP) duration to 90% repolarization (APD90), lower AP plateau potential and peak ICa-L current densities (Pcalpain 1 was increased in the adult and the aged groups with AF (Pcalpain 1. The general pathophysiological alterations in normal aged atria may therefore produce a substrate that is conducive to AF.

  20. A Versatile Imaging and Therapeutic Platform Based on Dual-Band Luminescent Lanthanide Nanoparticles toward Tumor Metastasis Inhibition.

    Science.gov (United States)

    Li, Yang; Tang, Jinglong; Pan, Dong-Xu; Sun, Ling-Dong; Chen, Chunying; Liu, Ying; Wang, Ye-Fu; Shi, Shuo; Yan, Chun-Hua

    2016-02-23

    Upconversion (UC) luminescent lanthanide nanoparticles (LNPs) are expected to play an important role in imaging and photodynamic therapy (PDT) in vitro and in vivo. However, with the absorption of UC emissions by photosensitizers (PSs) to generate singlet oxygen ((1)O2) for PDT, the imaging signals from LNPs are significantly weakened. It is important to activate another imaging route to track the location of the LNPs during PDT process. In this work, Nd(3+)-sensitized LNPs with dual-band visible and near-infrared (NIR) emissions under single 808 nm excitation were reported to address this issue. The UC emissions in green could trigger covalently linked rose bengal (RB) molecules for efficient PDT, and NIR emissions deriving from Yb(3+) and magnetic resonance imaging (MRI) were used for imaging simultaneously. Notably, the designed therapeutic platform could further effectively avoid the overheating effect induced by the laser irradiation, due to the minimized absorption of biological media at around 808 nm. TdT-mediated dUTP nick end labeling (TUNEL) assay showed serious cell apoptosis in the tumor after PDT for 2 weeks, leading to an effective tumor inhibition rate of 67%. Benefit from the PDT, the tumor growth-induced liver and spleen burdens were largely attenuated, and the liver injury was also alleviated. More importantly, pulmonary and hepatic tumor metastases were significantly reduced after PDT. The Nd(3+)-sensitized LNPs provide a multifunctional nanoplatform for NIR light-assisted PDT with minimized heating effect and an effective inhibition of tumor growth and metastasis.

  1. Apoptosis Resistance in Endometriosis

    Directory of Open Access Journals (Sweden)

    Liselotte Mettler

    2011-08-01

    Full Text Available Introduction: In a cytological analysis of endometriotic lesions neither granulocytes nor cytotoxic T-cells appear in an appreciable number. Based on this observation we aimed to know, whether programmed cell death plays an essential role in the destruction of dystopic endometrium. Disturbances of the physiological mechanisms of apoptosis, a persistence of endometrial tissue could explain the disease. Another aspect of this consideration is the proliferation competence of the dystopic mucous membrane. Methods: Endometriotic lesions of 15 patients were examined through a combined measurement of apoptosis activity with the TUNEL technique (terminal deoxyribosyltransferase mediated dUTP Nick End Labeling and the proliferation activity (with the help of the Ki-67-Antigens using the monoclonal antibody Ki-S5. Results: Twelve out of 15 women studied showed a positive apoptotic activity of 3-47% with a proliferation activity of 2-25% of epithelial cells. Therefore we concluded that the persistence of dystopic endometrium requires proliferative epithelial cells from middle to lower endometrial layers. Conclusion: A dystopia misalignment of the epithelia of the upper layers of the functionalism can be rapidly eliminated by apoptotic procedures.

  2. Andrographis paniculata extract induced apoptosis of adenocarcinoma mammae in C3H mice

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    Nugrahaningsih

    2013-08-01

    Full Text Available BACKGROUND Apoptosis plays an important role in tumorigenesis. Induction of apoptosis is a strategy for developing cancer therapy. In vitro study found that andrographolide isolated from Andrographis paniculata has anticancer activity by an apoptotic mechanism in cancer cell lines. The aim of the present study was to prove the effect of Andrographis paniculata extract administered orally on apoptosis of mammary adenocarcinoma in C3H mice. METHODS This study was of post test randomized control group design. Twenty four C3H mice with transplanted mammary adenocarcinomas were divided into four groups. To three groups Andrographis paniculata extract was administered orally for 14 days, at doses of 5, 10 and 15 mg/day, respectively, whereas to the control group no Andrographis paniculata extract was administered. On day 15 the mice were terminated. The mammary adenocarcinomas were examined by the terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL method. The values of the apoptotic index were expressed as mean±SD and analyzed using Anova and Pearson’s correlation test. RESULTS The mean apoptotic index values differed significantly among the experimental groups (p=0.001. The highest value was found in the group receiving Andrographis paniculata extract 15 mg/day, while the lowest was in the control group, the values being significantly correlated (r=0.974. CONCLUSIONS Oral administration of Andrographis paniculata extract induced apoptosis in C3H mice with mammary adenocarcinoma

  3. Effect of green light spectra on the reduction of retinal damage and stress in goldfish, Carassius auratus

    Energy Technology Data Exchange (ETDEWEB)

    Song, Jin Ah; Kim, Na Na; Choi, Young Jae; Choi, Cheol Young, E-mail: choic@kmou.ac.kr

    2016-07-22

    We investigated the effect of light spectra on retinal damage and stress in goldfish using green (530 nm) and red (620 nm) light emitting diodes (LEDs) at three intensities each (0.5, 1.0, and 1.5 W/m{sup 2}). We measured the change in the levels of plasma cortisol and H{sub 2}O{sub 2} and expression and levels of caspase-3. The apoptotic response of green and red LED spectra was assessed using the terminal transferase dUTP nick end labeling (TUNEL) assay. Stress indicator (cortisol and H{sub 2}O{sub 2}) and apoptosis-related genes (caspase-3) decreased in green light, but increased in red light with higher light intensities over time. The TUNEL assay revealed that more apoptotic cells were detected in outer nuclear layers after exposure to red LED over time with the increase in light intensity, than the other spectra. These results indicate that green light efficiently reduces retinal damage and stress, whereas red light induces it. Therefore, red light-induced retina damage may induce apoptosis in goldfish retina. -- Highlights: •Green light efficiently reduces retinal damage and stress. •Green spectra reduce caspase production and apoptosis. •Red light-induced retina damage may induce apoptosis in goldfish retina. •The retina of goldfish recognizes green spectra as a stable environment.

  4. Therapeutic effects of microbubble added to combined high-intensity focused ultrasound and chemotherapy in a pancreatic cancer xenograft model

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mi Hye [Dept. of Radiology, Konkuk University Medical Center, Seoul (Korea, Republic of); Lee, Jae Young; Kim, Bo Ram; Park, Eun Joo; Kim, Hoe Suk; Han, Joon Koo [Dept. of Radiology, Seoul National University Hospital, Seoul (Korea, Republic of); Kim, Hae Ri [Dept. of Pre-Dentistry, Gangneung-Wonju National University College of Dentistry, Gangneung (Korea, Republic of); Choi, Byung Ihn [Dept. of Radiology, Chung-Ang University Hospital, Seoul (Korea, Republic of)

    2016-09-15

    To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.

  5. Effect of Wenxin Granule on Ventricular Remodeling and Myocardial Apoptosis in Rats with Myocardial Infarction

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    Aiming Wu

    2013-01-01

    Full Text Available Aim. To determine the effect of a Chinese herbal compound named Wenxin Granule on ventricular remodeling and myocardial apoptosis in rats with myocardial infarction (MI. Methods. Male Sprague-Dawley (SD rats were randomly divided into four groups: the control group, the model group, the metoprolol group, and the Wenxin Granule group (WXKL group with sample size (n of 7 rats in each group. An MI model was established in all rats by occlusion of the left anterior descending coronary artery (the control group was without occlusion. Wenxin Granule (1.35 g/kg/day, metoprolol (12 mg/kg/day, and distilled water (5 mL/kg/day for the control and model groups were administered orally for 4 weeks. Ultrasonic echocardiography was used to examine cardiac structural and functional parameters. Myocardial histopathological changes were observed using haematoxylin and eosin (H&E dyeing. Myocardial apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL staining. Serum angiotensin II (Ang II concentration was measured using the enzyme-linked immunosorbent assay (ELISA. Results. It was found that Wenxin Granule could partially reverse ventricular remodeling, improve heart function, alleviate the histopathological damage, inhibit myocardial apoptosis, and reduce Ang II concentration in rats with MI. Conclusions. The results of the current study suggest that Wenxin Granule may be a potential alternative and complementary medicine for the treatment of MI.

  6. 5′-Adenosine Monophosphate-Induced Hypothermia Attenuates Brain Ischemia/Reperfusion Injury in a Rat Model by Inhibiting the Inflammatory Response

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    Yi-Feng Miao

    2015-01-01

    Full Text Available Hypothermia treatment is a promising therapeutic strategy for brain injury. We previously demonstrated that 5′-adenosine monophosphate (5′-AMP, a ribonucleic acid nucleotide, produces reversible deep hypothermia in rats when the ambient temperature is appropriately controlled. Thus, we hypothesized that 5′-AMP-induced hypothermia (AIH may attenuate brain ischemia/reperfusion injury. Transient cerebral ischemia was induced by using the middle cerebral artery occlusion (MCAO model in rats. Rats that underwent AIH treatment exhibited a significant reduction in neutrophil elastase infiltration into neuronal cells and matrix metalloproteinase 9 (MMP-9, interleukin-1 receptor (IL-1R, tumor necrosis factor receptor (TNFR, and Toll-like receptor (TLR protein expression in the infarcted area compared to euthermic controls. AIH treatment also decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL- positive neuronal cells. The overall infarct volume was significantly smaller in AIH-treated rats, and neurological function was improved. By contrast, rats with ischemic brain injury that were administered 5′-AMP without inducing hypothermia had ischemia/reperfusion injuries similar to those in euthermic controls. Thus, the neuroprotective effects of AIH were primarily related to hypothermia.

  7. Isorhamnetin attenuates atherosclerosis by inhibiting macrophage apoptosis via PI3K/AKT activation and HO-1 induction.

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    Yun Luo

    Full Text Available Isorhamnetin (Iso is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/- mice fed a high fat diet.Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions.In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction.

  8. Expression of MTLC gene in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Guang-Bin Qiu; Li-Guo Gong; Dong-Mei Hao; Zhi-Hong Zhen; Kai-Lai Sun

    2003-01-01

    AIM: To investigate the expression of c-myc target from laryngeal cancer cells (MTLC) gene in gastric carcinoma (GC)tissues and the effect of MTLC over-expression on gastric carcinoma cell line BGC823.METHODS: RT-PCR was performed to determine the expression of MTLC mRNA in GC and matched control tissues.BGC823 cells were transfected with an expression vector pcDNA3.1-MTLC by liposome and screened by G418. Growth of cells expressing MTLC was observed daily by manual counting. Apoptotic cells were determined by TdT-mediated dUTP nick-end labeling (TUNEL) assay.RESULTS: The expression of MTLC mRNAs was downregulated in 9(60%) of 15 cases of GC tissues. The growth rates of the BGC823 cells expressing MTLC were indistinguishable from that of control cells. A marked acceleration of apoptosis was observed in MTLC-expressing cells.CONCLUSION: MTLC was down-regulated in the majority of GC tissues and could promote apoptosis of GC cell lines,which suggests that MTLC may play an important role in the carcinogenesis of gastric carcinoma.

  9. Studies on the apoptosis of autonomic neuron in streptozotocin-induced diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Qinglin Lou; Rongwen Bian; Tao Peng; Xiaojun Ouyang; Hui Xia; Liubao Gu; Yongzhen Mo

    2006-01-01

    Objective: The aim of the study was to investigate apoptosis of the autonomic neuron in streptozotocin-induced diabetic rats, and observe the effect of intervention with nerve growth factor (NGF) on the apoptosis. Methods: A total of 29 male Sprague-Dawley rats were divided into three groups, i.e. normal control (NC, n = 12), untreated diabetic (DM, n =9) and diabetic treated with NGF daily of 500 μg/kg for 30 days(DM+NGF, n = 8). The diabetic rat models were produced by intraperitoneal injection with streptozotocin(65 mg/kg). Over 3 months since the diabetes were setup, the superior cervical sympathetic ganglions(SCG) and the celiac ganglions(CG) were removed and fixed with 10% paraformaldchyde. Apoptosis was measured using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling(TUNEL). Apoptotic index (AI) was calculated by computer image analysis system. Results: The AI of SCG and CG in DM and DM+NGF group were significant higher than those of NC group (P < 0.001) respectively. There was no difference of AI of SCG and CG between DM group and DM+NGF group (both P > 0.05). Conclusion: Neuron apoptosis may contribute to the pathophysiology of diabetic autonomic neuropathy and NGF can not prevent the apoptosis of autonomic neuron in diabetic rats.

  10. "The Role ofL-arginine in Control of Apoptosis in Preimplantation Mouse Embryos Cultured in High Glucose Media "

    Directory of Open Access Journals (Sweden)

    Mohammad Barbarestani

    2004-06-01

    Full Text Available Maternal hyperglycemia causes delay in early stages of embryonic growth and development, higher incidence of congenital malformations and spontaneous miscarriage compared with those of non-diabetic conditions. High glucosis tratogenicity seems to be related to reduction of Nitric Oxide production (NO in hyperglycemic condition. In order to test this hypothesis, 2-cell stage embryos of normal mice were cultured with high concentration of glucose (30mM and different concentrations of L-arginine (5,10,20 mM or L-NAME, an NO syntase (NOS inhibitor. In the end of culture, blastocysts were stained by by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL technique and apoptotic cells were detected by using a Fluorescence microscope. Finally the amount of nitrite in the cultured media was assayed by Griess method. The results indicated that high glucose reduces Nitric Oxide production by preimplantation embryos and increases apoptosis of embryonic cells, but 5-20mM of L-arginine significantly increases Nitric Oxide production and decreases apoptosis. On the contrary L-NAME significantly inhibits the development of pre-implantation embryos. In conclusion, this study indicated that reduced nitric oxide production in high glucosis condition is a main factor for embryonic damage, and supplementation of high glucose media with L-arginine has an important role in prevention of high glucosis embryotoxicity

  11. Valproic acid induces cutaneous wound healing in vivo and enhances keratinocyte motility.

    Directory of Open Access Journals (Sweden)

    Soung-Hoon Lee

    Full Text Available BACKGROUND: Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK signaling pathways. Valproic acid (VPA is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown. METHODS AND FINDINGS: We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA, collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase/Akt signaling pathways. CONCLUSIONS: VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes.

  12. Association between Up-regulation of Fas Ligand Expression and Apoptosis of Tumor-infiltrating Lymphocytes in Human Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    CHENG Bo

    2006-01-01

    In order to study the significance of FasL expression in immune escape of breast cancer,FasL protein expression and the number of tumor-infiltrating lymphocytes (TILs) in 40 specimens of breast cancer were detected by immunohistochemitry. The expression of FasL mRNA was measured by in situ hybridization in the consecutive tissue slices of 40 breast cancers respectively. By using terminal deoxynucleotidyl transferase-mediaed dUTP nick end labeling (TUNEL), apoptotic cells were detected in 40 specimens of breast cancer. The expression of FasL was detected in all 40 specimens to varying degrees. In the consecutive tissue slices, the location of expression of FasL protein corresponded with that of FasL mRNA. In those with FasL extensive expression, the number of TILs was less (P<0.05), the apoptotic index (AI) of TILs was higher and the AI of tumor cells was lower (P<0.01) than those with FasL weak expression respectively. The AI of TILs was correlated with that of tumor cells (r=-0.629, P<0.01). In conclusion, breast cancer cells can induce the apoptosis of TILs through the expression of FasL, which can counterattack the immune system. This may be a mechanism of immune evasion in breast cancer.

  13. Antileukemic effect of zerumbone-loaded nanostructured lipid carrier in WEHI-3B cell-induced murine leukemia model.

    Science.gov (United States)

    Rahman, Heshu Sulaiman; Rasedee, Abdullah; How, Chee Wun; Zeenathul, Nazariah Allaudin; Chartrand, Max Stanley; Yeap, Swee Keong; Abdul, Ahmad Bustamam; Tan, Sheau Wei; Othman, Hemn Hassan; Ajdari, Zahra; Namvar, Farideh; Arulselvan, Palanisamy; Fakurazi, Sharida; Mehrbod, Parvaneh; Daneshvar, Nasibeh; Begum, Hasina

    2015-01-01

    Cancer nanotherapy is progressing rapidly with the introduction of many innovative drug delivery systems to replace conventional therapy. Although the antitumor activity of zerumbone (ZER) has been reported, there has been no information available on the effect of ZER-loaded nanostructured lipid carrier (NLC) (ZER-NLC) on murine leukemia cells. In this study, the in vitro and in vivo effects of ZER-NLC on murine leukemia induced with WEHI-3B cells were investigated. The results from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Hoechst 33342, Annexin V, cell cycle, and caspase activity assays showed that the growth of leukemia cells in vitro was inhibited by ZER-NLC. In addition, outcomes of histopathology, transmission electron microscopy, and Tdt-mediated dUTP nick-end labeling analyses revealed that the number of leukemia cells in the spleen of BALB/c leukemia mice significantly decreased after 4 weeks of oral treatment with various doses of ZER-NLC. Western blotting and reverse-transcription quantitative polymerase chain reaction assays confirmed the antileukemia effects of ZER-NLC. In conclusion, ZER-NLC was shown to induce a mitochondrial-dependent apoptotic pathway in murine leukemia. Loading of ZER in NLC did not compromise the anticancer effect of the compound, suggesting ZER-NLC as a promising and effective delivery system for treatment of cancers.

  14. Downregulation of Endogenous Hydrogen Sulfide Pathway Is Involved in Mitochondrion-Related Endothelial Cell Apoptosis Induced by High Salt

    Directory of Open Access Journals (Sweden)

    Yanfang Zong

    2015-01-01

    Full Text Available Background. The study aimed to investigate whether endogenous H2S pathway was involved in high-salt-stimulated mitochondria-related vascular endothelial cell (VEC apoptosis. Methods. Cultured human umbilical vein endothelial cells (HUVECs were used in the study. H2S content in the supernatant was detected. Western blot was used to detect expression of cystathionine gamma-lyase (CSE, cleaved-caspase-3, and mitochondrial and cytosolic cytochrome c (cytc. Fluorescent probes were used to quantitatively detect superoxide anion generation and measure the in situ superoxide anion generation in HUVEC. Mitochondrial membrane pore opening, mitochondrial membrane potential, and caspase-9 activities were measured. The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL methods. Results. High-salt treatment downregulated the endogenous VEC H2S/CSE pathway, in association with increased generation of oxygen free radicals, decreased mitochondrial membrane potential, enhanced the opening of mitochondrial membrane permeability transition pore and leakage of mitochondrial cytc, activated cytoplasmic caspase-9 and caspase-3 and subsequently induced VEC apoptosis. However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt. Conclusion. H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult. The protective mechanisms for VEC damage might involve inhibiting oxidative stress and protecting mitochondrial injury.

  15. CD163/Hemoglobin Oxygenase-1 Pathway Regulates Inflammation in Hematoma Surrounding Tissues after Intracerebral Hemorrhage.

    Science.gov (United States)

    Liu, BaoHua; Hu, BeiLei; Shao, ShengMin; Wu, Wei; Fan, LiuBo; Bai, GuangHui; Shang, Ping; Wang, XiaoTong

    2015-12-01

    The aim of the present study was to investigate changes in the expression of CD163 and hemoglobin oxygenase-1 (HO-1) in brain tissue surrounding hematomas after intracerebral hemorrhage (ICH), and correlations with other factors. Brain tissues in the close surrounding of ICH hematomas (n = 27, ICH group) were collected at 6 hours or less, 6-24 hours, 24-72 hours, and more than 72 hours after bleeding onset, and more distant tissues (n = 12, control group) were histologically analyzed with hematoxylin and eosin staining and transmission electron microscopy. Interleukin (IL)-1, IL-10, and tumor necrosis factor-alpha, as well as the expression of CD163 and HO-1, were assessed using immunochemistry, Western blotting, and reverse transcription-polymerase chain reaction. Apoptosis rates were determined with terminal deoxynucleotidyl transferase dUTP nick end labeling assays. The expressions of the inflammatory cytokines IL-1 and tumor necrosis factor-alpha were increased at 6-24 hours (P CD163 and HO-1 expressions gradually increased from 6 to 24 hours to peaks at more than 72 hours after ICH onset (P CD163 and HO-1 expressions reached peaks and inflammatory cytokine expressions dropped. Copyright © 2015 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  16. Protective Effect of N-Acetylserotonin against Acute Hepatic Ischemia-Reperfusion Injury in Mice

    Directory of Open Access Journals (Sweden)

    Jiying Jiang

    2013-08-01

    Full Text Available The purpose of this study was to investigate the possible protective effect of N-acetylserotonin (NAS against acute hepatic ischemia-reperfusion (I/R injury in mice. Adult male mice were randomly divided into three groups: sham, I/R, and I/R + NAS. The hepatic I/R injury model was generated by clamping the hepatic artery, portal vein, and common bile duct with a microvascular bulldog clamp for 30 min, and then removing the clamp and allowing reperfusion for 6 h. Morphologic changes and hepatocyte apoptosis were evaluated by hematoxylin-eosin (HE and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining, respectively. Activated caspase-3 expression was evaluated by immunohistochemistry and Western blot. The activation of aspartate aminotransferase (AST, malondialdehyde (MDA, and superoxide dismutase (SOD was evaluated by enzyme-linked immunosorbent assay (ELISA. The data show that NAS rescued hepatocyte morphological damage and dysfunction, decreased the number of apoptotic hepatocytes, and reduced caspase-3 activation. Our work demonstrates that NAS ameliorates hepatic IR injury.

  17. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    徐昌杰; 陈昆松; FERGUSONIanB

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple celi death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  18. Programmed cell death features in apple suspension cells under low oxygen culture.

    Science.gov (United States)

    Xu, Chang-jie; Chen, Kun-song; Ferguson, Ian B

    2004-02-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  19. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    XU Chang-jie(徐昌杰); CHEN Kun-song(陈昆松); FERGUSON Ian B.

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  20. Attenuated Cardiac Mitochondrial-Dependent Apoptotic Effects by Li-Fu Formula in Hamsters Fed with a Hypercholesterol Diet

    Directory of Open Access Journals (Sweden)

    Wei-Wen Kuo

    2011-01-01

    Full Text Available Apoptosis involves in the pathogenesis of various cardiac abnormalities. This study intends to evaluate the effects of Li-Fu formula on cardiac apoptosis induced by hyper-cholesterol diet. Twenty-four male Golden Syrian hamsters were randomly divided into Control, Cholesterol and Li-Fu formula groups. Histopathological analysis, western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assays were performed to measure the effects of Li-Fu formula on left ventricle. Significantly reduced TUNEL-positive cells and mitochondria- dependent apoptosis were observed in the left ventricle of hamsters from Li-Fu formula group compared to the Cholesterol group. Additionally, induced cardiac insulin like growth factor I receptor (IGFIR-dependent survival pathway was detected in the Li-Fu formula group compared to the Cholesterol group. Besides, minor fibrosis, increased collagen deposition, and myofibril disarray was detected in the Cholesterol group, whereas the reductions of collagen deposition and myofibril disarray were observed in the Li-Fu formula group. This study demonstrated that Li-Fu formula not only reduced the mitochondria-dependent apoptosis and fibrosis, but also enhanced the IGF-I survival pathway in the left ventricle from high cholesterol-fed hamsters. We suggest the protective effects of Li-Fu formula on cardiac apoptosis and therapeutic potentials against cardiovascular disease.

  1. Isomenthone protects human dermal fibroblasts from TNF-α-induced death possibly by preventing activation of JNK and p38 MAPK.

    Science.gov (United States)

    Jung, Eunsun; Byun, Sangyo; Kim, Seungbeom; Kim, Moohan; Park, Deokhoon; Lee, Jongsung

    2012-10-01

    Cell death evoked by tumor necrosis factor-α (TNF-α) is regulated by the TNF-α receptor-associated death domain containing protein, which interacts with and activates apoptotic proteases triggering cell death. c-Jun N-terminal kinase (JNK) and p38 MAPK, induce the apoptotic program and are indispensible early elements in stress-induced apoptosis that control the release of cytochrome c. Isomenthone is a constituent of the essential oil of Mentha arvensis L. and is used as a fragrance and flavor in the cosmetic, drug, and food industries. In this study, we investigated the protective effects of isomenthone against TNF-α-induced cell death and its mechanism in human dermal fibroblasts. To understand the cytoprotective role of isomenthone, MTT and terminal deoxynucleotidyl transferase dUTP nick end labeling assays for cell viability and enzyme-linked immunosorbent assay analysis for the mechanistic study were performed. We found that isomenthone inhibited the TNF-α-mediated reduction in cell viability and inhibited the increase in apoptosis under a serum-free condition. Isomenthone also blocked the JNK and p38 MAPK pathways and downstream apoptotic events. These results indicate that isomenthone has the potential to protect fibroblasts against TNF-α-induced cell death under a serum-deprived condition by blocking activation of the JNK and p38 MAPK pathways and downstream apoptotic events.

  2. [Inhibition Function of Dominant-negative Mutant Gene Survivin-D53A to SPC-A1 Lung Adenocarcinoma Xenograft in Nude Mice Models].

    Science.gov (United States)

    Yu, Min; Peng, Xingchen; Lu, You; Huang, Meijuan

    2015-06-01

    Survivin-D53A (SVV-D53A) is a dominant-negative mutant survivin, which represents a potential promising target for cancer gene therapy. The present study was designed to determine whether SVV-D53A plasmid encapsuled by DOTAP: Chol liposome would have the anti-tumor activity against SPC-A1 lung adenocarcinoma, and to detect the possible mechanisms. In our experiment, SPC-A1 cells were transfected in vitro with SVV-D53A plasmid and examined for protein expression by Western blot, then flow cytometric analysis was used to detect apoptosis. SPC-A1 lung adenocarcinoma xenografts were established in vivo in the nude mice, which received the i. v. administrations of SVV-D53A plasmid/liposome complexes. After mice were sacrificed, the paraffin-embedded tumor tissue sections were used for proliferating cell nuclear antigen (PCNA) expression and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Compared with the control group, the mice treated with SVV-D53A plasmid had an obviously reduced tumor volume, with high level of apoptosis and decreased cell proliferation in tumor tissue. The research results proved that the administration of SVV-D53A plasmid resulted in significant inhibition of SPC-A1 cells both in vitro and in vivo. The functional mechanism is that the anti-tumor response causes and induces tumor cell apoptosis.

  3. A Taenia crassiceps metacestode factor enhances ovarian follicle atresia and oocyte degeneration in female mice.

    Science.gov (United States)

    Solano, S; Zepeda, N; Copitin, N; Fernandez, A M; Tato, P; Molinari, J L

    2015-01-01

    The histopathological effects of Taenia crassiceps infection or T. crassiceps metacestode factor inoculation on the mouse ovary were determined using six female mice in three groups: infected mice, mice inoculated with the metacestode factor and control mice. The control group was subcutaneously inoculated with healthy peritoneal fluid. The infected group was intraperitoneally inoculated with 40 T. crassiceps metacestodes, and the metacestode factor group was subcutaneously inoculated with T. crassiceps metacestode factor (MF). Light and electron microscopy and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling) assays revealed a significant increase in ovarian follicular atresia (predominantly in antral/preovulatory stages of development), oocyte degeneration (P< 0.05), and a decrease in the amount of corpus luteum in follicles of mice infected and inoculated with MF compared with the control group. Significant abnormalities of the granulosa cells and oocytes of the primordial, primary and secondary ovarian follicles occurred in both treated mouse groups (P< 0.05) compared with no degeneration in the control group. These pathological changes in female mice either infected with T. crassiceps metacestodes or inoculated with T. crassiceps MF may have consequences for ovulation and fertility.

  4. Effects of ultrasound and ultrasound contrast agent on vascular tissue

    Directory of Open Access Journals (Sweden)

    Wood Steven C

    2012-07-01

    Full Text Available Abstract Background Ultrasound (US imaging can be enhanced using gas-filled microbubble contrast agents. Strong echo signals are induced at the tissue-gas interface following microbubble collapse. Applications include assessment of ventricular function and virtual histology. Aim While ultrasound and US contrast agents are widely used, their impact on the physiological response of vascular tissue to vasoactive agents has not been investigated in detail. Methods and results In the present study, rat dorsal aortas were treated with US via a clinical imaging transducer in the presence or absence of the US contrast agent, Optison. Aortas treated with both US and Optison were unable to contract in response to phenylephrine or to relax in the presence of acetylcholine. Histology of the arteries was unremarkable. When the treated aortas were stained for endothelial markers, a distinct loss of endothelium was observed. Importantly, terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL staining of treated aortas demonstrated incipient apoptosis in the endothelium. Conclusions Taken together, these ex vivo results suggest that the combination of US and Optison may alter arterial integrity and promote vascular injury; however, the in vivo interaction of Optison and ultrasound remains an open question.

  5. HIV-1gp120 induces neuronal apoptosis through enhancement of 4-aminopyridine-senstive outward K+ currents.

    Directory of Open Access Journals (Sweden)

    Lina Chen

    Full Text Available Human immunodeficiency virus type 1 (HIV-1-associated dementia (HAD usually occurs late in the course of HIV-1 infection and the mechanisms underlying HAD pathogenesis are not well understood. Accumulating evidence indicates that neuronal voltage-gated potassium (Kv channels play an important role in memory processes and acquired neuronal channelopathies in HAD. To examine whether Kv channels are involved in HIV-1-associated neuronal injury, we studied the effects of HIV-1 glycoprotein 120 (gp120 on outward K+ currents in rat cortical neuronal cultures using whole-cell patch techniques. Exposure of cortical neurons to gp120 produced a dose-dependent enhancement of A-type transient outward K+ currents (IA. The gp120-induced increase of IA was attenuated by T140, a specific antagonist for chemokine receptor CXCR4, suggesting gp120 enhancement of neuronal IA via CXCR4. Pretreatment of neuronal cultures with a protein kinase C (PKC inhibitor, GF109203X, inhibited the gp120-induced increase of IA. Biological significance of gp120 enhancement of IA was demonstrated by experimental results showing that gp120-induced neuronal apoptosis, as detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay and caspase-3 staining, was attenuated by either an IA blocker 4-aminopyridine or a specific CXCR4 antagonist T140. Taken together, these results suggest that gp120 may induce caspase-3 dependent neuronal apoptosis by enhancing IA via CXCR4-PKC signaling.

  6. Caspase-3-dependent apoptosis of citreamicin ε-induced heLa iells Is associated with reactive oxygen species generation

    KAUST Repository

    Liu, Lingli

    2013-07-15

    Citreamicins, members of the polycyclic xanthone family, are promising antitumor agents that are produced by Streptomyces species. Two diastereomers, citreamicin ε A (1) and B (2), were isolated from a marine-derived Streptomyces species. The relative configurations of these two diastereomers were determined using NMR spectroscopy and successful crystallization of citreamicin ε A (1). Both diastereomers showed potent cytotoxic activity against HeLa (cervical cancer) and HepG2 (hepatic carcinoma) cells with IC 50 values ranging from 30 to 100 nM. The terminal deoxynucleotidyl transferase dUTP nick-end labeling assay confirmed that citreamicin ε A (1) induced cellular apoptosis, and Western blot analysis showed that apoptosis occurred via activation of caspase-3. The 2,7-dichlorofluorescein diacetate assay indicated that citreamicin ε substantially increased the intracellular concentration of reactive oxygen species (ROS). To confirm the hypothesis that citreamicin ε induced apoptosis through an increase in the intracellular ROS concentration, the oxidized products, oxicitreamicin ε A (3) and B (4), were obtained from a one-step reaction catalyzed by Ag 2O. These products, with a reduced capacity to increase the intracellular ROS concentration, exhibited a significantly weakened cytotoxicity in both HeLa and HepG2 cells compared with that of citreamicin ε A (1) and B (2). © 2013 American Chemical Society.

  7. Premature Tapetum Degeneration: a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

    Institute of Scientific and Technical Information of China (English)

    Yinlian Shi; Sha Zhao; Jialing Yao

    2009-01-01

    Photoperiod-sensitive genic male-sterile (PSGMS) rice (Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT-mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.

  8. High clusterin expression correlates with a poor outcome in stage II colorectal cancers.

    LENUS (Irish Health Repository)

    Kevans, David

    2012-02-01

    The role of clusterin in tumor growth and progression remains unclear. Overexpression of cytoplasmic clusterin has been studied in aggressive colon tumors; however, no correlation between clusterin expression and survival in colorectal cancer has been identified to date. We assessed levels of clusterin expression in a group of stage II colorectal cancer patients to assess its utility as a prognostic marker. The study included 251 patients with stage II colorectal cancer. Tissue microarrays were constructed and immunohistochemistry done and correlated with clinical features and long term outcome. Dual immunofluorescence and confocal microscopy were used with terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling probes and clusterin antibody to assess the degree of co localization. Percentage epithelial cytoplasmic staining was higher in tumor compared with nonadjacent normal mucosa (P < 0.001). Within the stromal compartment, percentage cytoplamic staining and intensity was lower in tumor tissue compared with normal nonadjacent mucosa (P < or = 0.001). Survival was significantly associated with percentage epithelial cytoplasmic staining (P < 0.001), epithelial cytoplasmic staining intensity (P < 0.001), percentage stromal cytoplasmic staining (P = 0.002), and stromal cytoplasmic staining intensity (P < 0.001). Clusterin levels are associated with poor survival in stage II colorectal cancer.

  9. Low-dose radiation modifies skin response to acute gamma-rays and protons.

    Science.gov (United States)

    Mao, Xiao Wen; Pecaut, Michael J; Cao, Jeffrey D; Moldovan, Maria; Gridley, Daila S

    2013-01-01

    The goal of the present study was to obtain pilot data on the effects of protracted low-dose/low-dose-rate (LDR) γ-rays on the skin, both with and without acute gamma or proton irradiation (IR). Six groups of C57BL/6 mice were examined: a) 0 Gy control, b) LDR, c) Gamma, d) LDR+Gamma, e) Proton, and f) LDR+Proton. LDR radiation was delivered to a total dose of 0.01 Gy (0.03 cGy/h), whereas the Gamma and Proton groups received 2 Gy (0.9 Gy/min and 1.0 Gy/min, respectively). Assays were performed 56 days after exposure. Skin samples from all irradiated groups had activated caspase-3, indicative of apoptosis. The significant (pGamma and Proton groups were not present when LDR pre-exposure was included. However, the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay for DNA fragmentation and histological examination of hematoxylin and eosin-stained sections revealed no significant differences among groups, regardless of radiation regimen. The data demonstrate that caspase-3 activation initially triggered by both forms of acute radiation was greatly elevated in the skin nearly two months after whole-body exposure. In addition, LDR γ-ray priming ameliorated this response.

  10. Farnesol induces apoptosis and oxidative stress in the fungal pathogen Penicillium expansum.

    Science.gov (United States)

    Liu, Pu; Luo, Li; Guo, Junhong; Liu, Huimin; Wang, Baoquan; Deng, Boxun; Long, Chao-an; Cheng, Yunjiang

    2010-01-01

    This study was conducted to evaluate the effect of farnesol (FOH) on the growth of P. expansum. The viability of P. expansum cells was determined by counting the colony forming units (CFU) after each FOH treatment. Morphological changes of FOH-treated fungal cells were analyzed by staining with Hoechst 33258, TUNEL (terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling), Annexin-V FITC and the oxidant-sensitive probe H2DCFDA (dichlorodihydro-fluorescein diacetate). FOH strongly inhibited the growth of hyphae. The hyphal cells showed the hallmarks of apoptosis including chromatin condensation, DNA fragmentation, phosphatidylserine (PS) externalization, caspases activation, intracellular reactive oxygen species (ROS) generation but without nucleosomal ladder production. The abnormal cellular ultrastructure observed by transmission electron microscope (TEM) indicated that disintegration of cellular ultrastructure (especially for mitochondria) was linked to FOH-induced cell death. Taken together we demonstrated that FOH inhibits the growth of P. expansum and promotes apoptosis via activation of metacaspases, production of ROS and disintegration of cellular ultrastructure.

  11. Bulky DNA adducts in human sperm associated with semen parameters and sperm DNA fragmentation in infertile men: a cross-sectional study

    Science.gov (United States)

    2013-01-01

    Background DNA adducts are widely used marker of DNA damage induced by environmental pollutants. The present study was designed to explore whether sperm polycyclic aromatic hydrocarbon-DNA adducts were associated with sperm DNA integrity and semen quality. Methods A total of 433 Han Chinese men were recruited from an infertility clinic. Immunofluorescence was applied to analyze sperm PAH-DNA adducts. Sperm DNA fragmentation was detected by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end labelling (TUNEL) assay. Results After adjustment for potential confounders using linear regression, sperm PAH-DNA adducts were negatively associated with sperm concentration, total sperm count, sperm motility, and curvilinear velocity (VCL). In addition, a positive relationship between sperm PAH-DNA adducts and sperm DNA fragmentation was found. Conclusions Our findings suggested an inverse association between sperm PAH-DNA adducts and semen quality, and provided the first epidemiologic evidence of an adverse effect of PAH-DNA adducts on sperm DNA integrity. PMID:24073787

  12. TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes: the effect of microorganisms within human follicular fluid collected during IVF cycles.

    Science.gov (United States)

    Pelzer, Elise S; Harris, Jessica E; Allan, John A; Waterhouse, Mary A; Ross, Tara; Beagley, Kenneth W; Knox, Christine L

    2013-09-01

    Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours' incubation within human follicular fluids (n=5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (DNA fragmentation in mouse oocytes following 12h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism.

    Science.gov (United States)

    Ruvolo, Giovanni; Roccheri, Maria Carmela; Brucculeri, Anna Maria; Longobardi, Salvatore; Cittadini, Ettore; Bosco, Liana

    2013-04-01

    An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %. Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .

  14. Evaluation of sperm head shape at high magnification revealed correlation of sperm DNA fragmentation with aberrant head ellipticity and angularity.

    Science.gov (United States)

    Utsuno, Hiroki; Oka, Kenji; Yamamoto, Ayako; Shiozawa, Tanri

    2013-05-01

    To test for an association between DNA fragmentation and head shape at high magnification in fresh motile spermatozoa. Observational study. Academic tertiary care center. A total of 60 men in our assisted reproductive program. Quantifying sperm head shape using elliptic Fourier analysis, and detecting DNA fragmentation by use of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Correlation between percentage of spermatozoa with abnormal head shape and percentage of DNA fragmentation. Elliptic Fourier analysis decomposed sperm head shapes into four quantitative parameters: ellipticity, anteroposterior (AP) symmetry, lateral symmetry, and angularity. The DNA fragmentation was statistically significantly correlated with abnormal angularity, and moderately with abnormal ellipticity but not with abnormal AP symmetry or lateral symmetry. Forward stepwise multiple logistic regression analysis revealed a statistically significantly higher percentage of DNA fragmentation in spermatozoa with abnormal ellipticity and abnormal angularity than in spermatozoa with normal-shaped head (6.1% and 5.4% vs. 2.8%). Spermatozoa with large nuclear vacuoles also correlated with sperm DNA fragmentation, and had a statistically significantly higher percentage of DNA fragmentation (4.7%). Among the morphologic features of the sperm head, abnormal ellipticity, angularity, and large nuclear vacuoles are associated with DNA fragmentation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  15. Chromosomal aneuploidies and DNA fragmentation of human spermatozoa from patients exposed to perfluorinated compounds.

    Science.gov (United States)

    Governini, L; Guerranti, C; De Leo, V; Boschi, L; Luddi, A; Gori, M; Orvieto, R; Piomboni, P

    2015-11-01

    This study investigated chromosomal aneuploidies and DNA damage in spermatozoa from male patients contaminated by perfluorinated compounds (PFCs) in whole blood and seminal plasma. Sperm aneuploidy and diploidy rate for chromosomes 18, X and Y were evaluated by FISH; sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling technique coupled to flow cytometry. Our results indicated that PFC contamination was present in 58% of subjects included in the study. A significant increase in alterations of sperm parameters was observed in PFC-positive subjects compared to PFC-negative subjects. As regards the sperm aneuploidy, both disomy and diploidy rates resulted significantly increased in subjects positive for PFC contamination compared to PFC-negative samples. In addition, sperm DNA fragmentation index resulted significantly increased in PFC-contaminated subjects compared to PFC-non-contaminated subjects, with a significant increased level of dimmer DNA fragmentation index. Our results clearly indicate that PFC contamination may detrimentally affect spermatogenesis, disturbing both meiotic segregation and DNA integrity. We could therefore suggest cautions to reduce or eliminate any contact with these compounds because the long-term effects of PFC accumulation in the body are not predictable. © 2014 Blackwell Verlag GmbH.

  16. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage.

    Science.gov (United States)

    Coughlan, Carol; Clarke, Helen; Cutting, Rachel; Saxton, Jane; Waite, Sarah; Ledger, William; Li, Tinchiu; Pacey, Allan A

    2015-01-01

    Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

  17. Mechanical processing of hyperviscous semen specimens can negatively affect sperm DNA fragmentation.

    Science.gov (United States)

    Kussler, Ana Paula S; Pimentel, Anita M; Alcoba, Diego D; Liu, Isabella P; Brum, Ilma Simoni; Capp, Edison; Corleta, Helena V E

    2014-04-01

    The present study compared the DNA fragmentation in human sperm samples with reduced, physiological, and increased viscosity in order to evaluate whether the process used to reduce viscosity (expulsion of semen through a needle and syringe) alters significantly sperm DNA fragmentation. The seminal parameters of semen samples from 123 patients were evaluated and classified according to their viscosity. Samples with increased viscosity were submitted to a process of expulsion of semen through a 10-mL syringe and an 18-gauge (18G) needle to reduce the seminal viscosity. The DNA fragmentation of all samples was analysed using TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick-end labelling assay); in samples with increased viscosity, the fragmentation was assessed before and after the process of expulsion with syringe and needle. There was no difference in DNA fragmentation between groups with different viscosity (P = 0.857). A significantly increase in sperm DNA fragmentation after expulsion of hyperviscous semen through the syringe was observed (P = 0.035). There was no difference in DNA fragmentation rate between samples with reduced, increased and physiological viscosities; however, the physical process of expulsion of semen through a syringe and needle increased sperm DNA fragmentation.

  18. Correlation between Gene Expression and Osteoarthritis Progression in Human

    Directory of Open Access Journals (Sweden)

    Leilei Zhong

    2016-07-01

    Full Text Available Osteoarthritis (OA is a multifactorial disease characterized by gradual degradation of joint cartilage. This study aimed to quantify major pathogenetic factors during OA progression in human cartilage. Cartilage specimens were isolated from OA patients and scored 0–5 according to the Osteoarthritis Research Society International (OARSI guidelines. Protein and gene expressions were measured by immunohistochemistry and qPCR, respectively. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assays were used to detect apoptotic cells. Cartilage degeneration in OA is a gradual progress accompanied with gradual loss of collagen type II and a gradual decrease in mRNA expression of SOX9, ACAN and COL2A1. Expression of WNT antagonists DKK1 and FRZB was lost, while hypertrophic markers (RUNX2, COL10A1 and IHH increased during OA progression. Moreover, DKK1 and FRZB negatively correlated with OA grading, while RUNX2 and IHH showed a significantly positive correlation with OA grading. The number of apoptotic cells was increased with the severity of OA. Taken together, our results suggested that genetic profiling of the gene expression could be used as markers for staging OA at the molecular level. This helps to understand the molecular pathology of OA and may lead to the development of therapies based on OA stage.

  19. Protective effects of astragalus extract against intermittent hypoxia-induced hippocampal neurons impairment in rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qiang; GAO Wen-yuan; ZHANG Yun; CHEN Bao-yun; CHEN Zhe; ZHANG Wei-san; MAN Shu-li

    2013-01-01

    Background Intermittent hypoxia is the main pathophysiological cause of the obstructive sleep apnea syndrome.Astragalus shows improvement of spatial learning and memory abilities under intermittent hypoxia.Our study aimed to investigate the protective effect of astragalus against intermittent hypoxia induced-hippocampal neurons impairment in rats and lay the theoretical foundation for the sleep apnea improvement in cognitive function by astragalus.Methods Male Wistar rats were divided into 4 groups:blank control group,normoxia group,intermittent hypoxia group and astragalus treated intermittent hypoxia group.After 6-week treatment,apoptosis of neurons was evaluated by terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay.Furthermore,the expression of HIF-1a was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) at the mRNA level as well as by immunohistochemistry (IHC) and Western blotting at the protein level.Results HPLC analysis indicated that astragaloside Ⅳ,astragaloside Ⅱ and astragaloside Ⅰ were the main compounds in astragals extract.Astragalus extract reduced the apoptosis of hippocampal neurons (P <0.05) and decreased the expression of HIF-1a at both the mRNA and protein levels in hippocampus compared with non-treated groups (P <0.05).Conclusion Astragalus protects against intermittent hypoxia-induced hippocampal neurons impairment in rats.

  20. Partial deletion of argininosuccinate synthase protects from pyrazole plus lipopolysaccharide-induced liver injury by decreasing nitrosative stress.

    Science.gov (United States)

    Lu, Yongke; Leung, Tung Ming; Ward, Stephen C; Nieto, Natalia

    2012-02-01

    Argininosuccinate synthase (ASS) is the rate-limiting enzyme in the urea cycle. Along with nitric oxide synthase (NOS)-2, ASS endows cells with the L-citrulline/nitric oxide (NO·) salvage pathway to continually supply L-arginine from L-citrulline for sustained NO· generation. Because of the relevant role of NOS in liver injury, we hypothesized that downregulation of ASS could decrease the availability of intracellular substrate for NO· synthesis by NOS-2 and, hence, decrease liver damage. Previous work demonstrated that pyrazole plus LPS caused significant liver injury involving NO· generation and formation of 3-nitrotyrosine protein adducts; thus, wild-type (WT) and Ass+/- mice (Ass+/+ mice are lethal) were treated with pyrazole plus LPS, and markers of nitrosative stress, as well as liver injury, were analyzed. Partial ablation of Ass protected from pyrazole plus LPS-induced liver injury by decreasing nitrosative stress and hepatic and circulating TNFα. Moreover, apoptosis was prevented, since pyrazole plus LPS-treated Ass+/- mice showed decreased phosphorylation of JNK; increased MAPK phosphatase-1, which is known to deactivate JNK signaling; and lower cleaved caspase-3 than treated WT mice, and this was accompanied by less TdT-mediated dUTP nick end labeling-positive staining. Lastly, hepatic neutrophil accumulation was almost absent in pyrazole plus LPS-treated Ass+/- compared with WT mice. Partial Ass ablation prevents pyrazole plus LPS-mediated liver injury by reducing nitrosative stress, TNFα, apoptosis, and neutrophil infiltration.

  1. The newly synthesized 2-(3-hydroxy-5-methoxyphenyl)-6,7-methylenedioxyquinolin-4-one triggers cell apoptosis through induction of oxidative stress and upregulation of the p38 MAPK signaling pathway in HL-60 human leukemia cells.

    Science.gov (United States)

    Cheng, Yung-Yi; Yang, Jai-Sing; Tsai, Shih-Chang; Liaw, Chih-Chuang; Chung, Jing-Gung; Huang, Li-Jiau; Lee, Kuo-Hsiung; Lu, Chi-Cheng; Chien, Hsi-Cheng; Tsuzuki, Minoru; Kuo, Sheng-Chu

    2012-10-01

    The aim of the present study was to discover the signaling pathways associated with 2-(3-hydroxy-5-methoxy-phenyl)-6,7-methylenedioxyquinolin-4-one (YYK1)-induced apoptosis in HL-60 human leukemia cells. YYK1 induced cytotoxic effects, cell morphological changes, decreased the cell number and increased reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (ΔΨm) in HL-60 cells. YYK1-induced apoptosis was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results from colorimetric assays and western blot analysis indicated that activities of caspase-7/-3, caspase-8 and caspase-9 were increased in YYK1-treated HL-60 cells. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas/CD95, FasL and FADD), intrinsic related proteins (cytochrome c, Apaf-1, AIF and Endo G), the ratio of Bax/Bcl-2 and phosphorylated p38 MAPK were increased in HL-60 cells after YYK1 treatment. Cell apoptosis was significantly reduced after pre-treatment with N-acetylcysteine (NAC; a ROS scavenger) or diphenyleneiodonium chloride (DPI; a NADPH oxidase inhibitor). Blockage of p38 MAPK signaling by SB202190 abolished YYK1-induced Fas/CD95 upregulation and apoptosis in HL-60 cells. We conclude that YYK1 induces both of extrinsic and intrinsic apoptotic pathways via ROS-mediated activation of p38 MAPK signaling in HL-60 human leukemia cells in vitro.

  2. 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.

  3. Lychee Seed Saponins Improve Cognitive Function and Prevent Neuronal Injury via Inhibiting Neuronal Apoptosis in a Rat Model of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Xiuling Wang

    2017-02-01

    Full Text Available Lychee seed is a traditional Chinese medicine and possesses many activities, including hypoglycemia, liver protection, antioxidation, antivirus, and antitumor. However, its effect on neuroprotection is still unclear. The present study investigated the effects of lychee seed saponins (LSS on neuroprotection and associated mechanisms. We established a rat model of Alzheimer’s disease (AD by injecting Aβ25–35 into the lateral ventricle of rats and evaluated the effect of LSS on spatial learning and memory ability via the Morris water maze. Neuronal apoptosis was analyzed by hematoxylin and eosin stain and terminal deoxynucleotidyl transferase (Tdt-mediated dUTP nick-end labeling analysis, and mRNA expression of caspase-3 and protein expressions of Bax and Bcl-2 by reverse transcription-polymerase chain reaction (RT-PCR and Western blotting, respectively. The results showed that LSS remarkably improved cognitive function and alleviated neuronal injury by inhibiting apoptosis in the hippocampus of AD rats. Furthermore, the mRNA expression of caspase-3 and the protein expression of Bax were downregulated, while the protein expression of Bcl-2 and the ratio of Bcl-2/Bax were increased by LSS. We demonstrate that LSS significantly improves cognitive function and prevent neuronal injury in the AD rats via regulation of the apoptosis pathway. Therefore, LSS may be developed as a nutritional supplement and sold as a drug for AD prevention and/or treatment.

  4. A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing (137)Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin.

    Science.gov (United States)

    Grigaravicius, Paulius; Rapp, Alexander; Greulich, Karl Otto

    2009-03-01

    In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells. A somewhat more detailed inspection shows that the damage caused by 20 Gy ionizing radiation and by a single laser pulse of 10 microJ are comparable, while the damage caused by 12 microg/ml BLM depends highly on the individual cell. Taken together, this work provides a detailed view of DNA fragmentation induced by different treatments and allows comparing them to some extent, especially with respect to the neutral comet assay.

  5. Der allerschönste Krieg : [aus "Kõige ilusam sõda"¡ ("Der allerschönste Krieg")] : [luuletused] / Asko Künnap ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Künnap, Asko

    2006-01-01

    Sisu: Der allerschönste Krieg : Ich suchte mein nichtgelebtes Leben ; Wie fühlt sich's Seite an Seite ; So toll, dass wir kommen konnten ; Wie kommt man lebend aus Schweden zurück? ; Im Schlaf vereint ; Ruft die Spinnen - dass sie die Nacht weben ; Du hältst die Welt von mir fern ; Ruder mich zur Insel ; In den Augen des Mädchens aus dem Norden ; Der allerschönste Krieg ; Der Teufel im Sakko zu sechshundert Kronen. Orig.: Kõige ilusam sõda : Otsisin oma elamata jäänud elu ; Kuidas on kõrvuti olla ; Nii tore, et tulla saime ; Kuidas tulla elusana tagasi Rootsist? ; Unes ühes ; Kutsuge ämblikud - las punuvad öö ; Sina hoiad maailma minust ära ; Sõua mind saareni ; Ühe põhjamaa tüdruku silmades ; Kõige ilusam sõda ; Kurat kuuesajakroonises pintsakus

  6. Hledání identity v kontextu tzv. plantážnické paměti v povídkách Eudory Weltyové

    OpenAIRE

    Plicková, Michaela

    2015-01-01

    The present MA thesis discusses Eudora Welty's short fiction and the author's engagement with the plantation memory. The introductory chapter defines the concept of plantation memory as a flux of the normative plantation binaries, the plantation mythology obscuring the ante-bellum Southern reality, the linguistic and phenomenal evidence of the prevailing oppression, and the ability of the text and its creator to subvert the official narratives and to liberate the individuals' silenced voices....

  7. Theoretical study of mononuclear nickel(I), nickel(0), copper(i), and cobalt(I) dioxygen complexes: new insight into differences and similarities in geometry and bonding nature.

    Science.gov (United States)

    Chen, Yue; Sakaki, Shigeyoshi

    2013-11-18

    Geometries, bonding nature, and electronic structures of (N^N)Ni(O2) (N^N = β-diketiminate), its cobalt(I) and copper(I) analogues, and (Ph3P)2Ni(O2) were investigated by density functional theory (DFT) and multistate restricted active space multiconfigurational second-order perturbation (MS-RASPT2) methods. Only (N^N)Ni(O2) takes a C(S) symmetry structure, because of the pseudo-Jahn-Teller effect, while all other complexes take a C(2V) structure. The symmetry lowering in (N^N)Ni(O2) is induced by the presence of the singly occupied δ(d(xy)-π(x)*) orbital. In all of these complexes, significant superoxo (O2-) character is found from the occupation numbers of natural orbitals and the O-O π* bond order, which is independent of the number of d electrons and the oxidation state of metal center. However, this is not a typical superoxo species, because the spin density is not found on the O2 moiety, even in open-shell complexes, (N^N)Ni(O2) and (N^N)Co(O2). The M-O and O-O distances are considerably different from each other, despite the similar superoxo character. The M-O distance and the interaction energy between the metal and O2 moieties are determined by the d(yz) orbital energy of the metal moiety taking the valence state. The binding energy of the O2 moiety is understood in terms of the d(yz) orbital energy in the valence state and the promotion energy of the metal moiety from the ground state to the valence state. Because of the participations of various charge transfer (CT) interactions between the metal and O2 moieties, neither the d(yz) orbital energy nor the electron population of the O2 moiety are clearly related to the O-O bond length. Here, the π bond order of the O2 moiety is proposed as a good measure for discussing the O-O bond length. Because the d electron configuration is different among these complexes, the CT interactions are different, leading to the differences in the π bond order and, hence, the O-O distance among these complexes. The reactivity of dioxygen complex is discussed with the d(yz) orbital energy.

  8. 含镍废水的隔膜电解法回收工艺研究%Study on the NickeI Wastewater Treatment by Anion Exchange Membrane Diaphragm EIectroIysis

    Institute of Scientific and Technical Information of China (English)

    葛明敏; 王琪; 朱炳龙; 童霏; 周全法

    2015-01-01

    以含有一定浓度硼酸的含镍电镀废水作为阴极液,2%的NaOH水溶液为阳极液,阴离子交换膜为隔膜材料组成隔膜电解装置,对含镍电镀废水进行了回收工艺研究。考察了电解时间、电流密度、pH值、温度及硼酸浓度对镍回收率的影响。结果表明,延长反应时间、提高电解温度、增加硼酸浓度有利于提高回收率。在电流密度为300 A/m2,pH=4,温度为35℃,硼酸浓度为25 g/L的最佳工艺条件下电解6 h,镍的回收率高达96.64%,所得镍为松枝状结构电解镍,纯度为94.69%。%The nickel-containing electroplating waste water has been treated by anion exchange membrane e-lectrolysis device. The effects of electrolysis time,current density,pH,temperature and concentration of boric acid on nickel recovery were studied in this paper. The results showed that the increasing of reaction time,e-lectrolysis temperature,the concentration of boric acid could improve the recovery rate. Optimization of the e-lectrolysis conditions were obtained as follows:electrolysis time of 6 h,a current density of 300A / m2,pH =4,a temperature of 35℃,a boric acid concentration of 25g/L. The pine - like electrolytic nickel was a-chieved,recovery rate of nickel was 96. 64%,purity of nickel was 94. 69 %.

  9. Apoptosis in human hepatocellular carcinoma by terminal deoxynucleotidyl transferase-mediate dUTP-FITC nick end labeling%TUNEL技术原位观察肝细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    李江; 王文亮; 王文勇; 刘斌; 王泊云

    1998-01-01

    目的采用TUNEL技术对福尔马林固定、石蜡包埋的肝组织和培养肝癌细胞进行染色,以探讨肝细胞凋亡的形态特征和分布.方法使用德国宝灵曼公司生产的原位末端标记试剂盒. 石蜡包埋组织经微波处理后,行常规TUNEL染色,观察凋亡细胞的形态和分布.结果 TUNEL阳性信号为黄绿色或黄色荧光,位于胞核,呈小圆形或环行. TUNEL阳性细胞大多在形态学上表现为凋亡的细胞. 对肝组织TUNEL LI的分析显示,肝癌组织中细胞凋亡指数(0.395)比肝硬变(0.075)和癌旁肝组织(0.105)要高,且癌组织恶性程度越高,这种凋亡就越显著.结论改进的TUNEL技术对福尔马林固定、石蜡包埋的难处理组织进行染色是一个有效的方法.

  10. Der allerschönste Krieg : [aus "Kõige ilusam sõda"¡ ("Der allerschönste Krieg")] : [luuletused] / Asko Künnap ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Künnap, Asko

    2006-01-01

    Sisu: Der allerschönste Krieg : Ich suchte mein nichtgelebtes Leben ; Wie fühlt sich's Seite an Seite ; So toll, dass wir kommen konnten ; Wie kommt man lebend aus Schweden zurück? ; Im Schlaf vereint ; Ruft die Spinnen - dass sie die Nacht weben ; Du hältst die Welt von mir fern ; Ruder mich zur Insel ; In den Augen des Mädchens aus dem Norden ; Der allerschönste Krieg ; Der Teufel im Sakko zu sechshundert Kronen. Orig.: Kõige ilusam sõda : Otsisin oma elamata jäänud elu ; Kuidas on kõrvuti olla ; Nii tore, et tulla saime ; Kuidas tulla elusana tagasi Rootsist? ; Unes ühes ; Kutsuge ämblikud - las punuvad öö ; Sina hoiad maailma minust ära ; Sõua mind saareni ; Ühe põhjamaa tüdruku silmades ; Kõige ilusam sõda ; Kurat kuuesajakroonises pintsakus

  11. Stille wird zu Farben : [aus "Vaikus saab värvideks" ("Stille wird zu Farben")] : [luuletused] / Jaan Kaplinski ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Kaplinski, Jaan, 1941-

    2006-01-01

    Sisu: Stille wird zu Farben ; Der letzte rote Apfel ; Wer allein bleibt ; Ruf nach dem Vorherigen ; Mit jedem Frühling ; So viele Namen ; In die Spuren ; Höher ; Es wird Licht ; Mit dem Sommerwrind um die Wette ; Ob sie noch hört. Orig.: Vaikus saab värvideks ; Viimane verev õun ; Üksi jääb ; Hüüab eelmist ; Iga kevadega ; Niipalju nimesid ; Astub tuule jälgedele ; Käib kõrgemalt ; Saab valguseks ; Suvetuulega võidu ; Kas kuuleb enam

  12. Stille wird zu Farben : [aus "Vaikus saab värvideks" ("Stille wird zu Farben")] : [luuletused] / Jaan Kaplinski ; tlk. Irja Grönholm ja Gisbert Jänicke

    Index Scriptorium Estoniae

    Kaplinski, Jaan, 1941-

    2006-01-01

    Sisu: Stille wird zu Farben ; Der letzte rote Apfel ; Wer allein bleibt ; Ruf nach dem Vorherigen ; Mit jedem Frühling ; So viele Namen ; In die Spuren ; Höher ; Es wird Licht ; Mit dem Sommerwrind um die Wette ; Ob sie noch hört. Orig.: Vaikus saab värvideks ; Viimane verev õun ; Üksi jääb ; Hüüab eelmist ; Iga kevadega ; Niipalju nimesid ; Astub tuule jälgedele ; Käib kõrgemalt ; Saab valguseks ; Suvetuulega võidu ; Kas kuuleb enam

  13. The synthetic inhibitor of Fibroblast Growth Factor Receptor PD166866 controls negatively the growth of tumor cells in culture

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    Castelli Mauro

    2009-12-01

    Full Text Available Abstract Background Many experimental data evidence that over-expression of various growth factors cause disorders in cell proliferation. The role of the Fibroblast Growth Factors (FGF in growth control is indisputable: in particular, FGF1 and its tyrosine kinase receptor (FGFR1 act through a very complex network of mechanisms and pathways. In this work we have evaluated the antiproliferative activity effect of PD166866, a synthetic molecule inhibiting the tyrosin kinase action of FGFR1. Methods Cells were routinely grown in Dulbecco Modified Eagle's medium supplemented with newborn serum and a penicillin-streptomycin mixture. Cell viability was evaluated by Mosmann assay and by trypan blue staining. DNA damage was assessed by in situ fluorescent staining with Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL assay. Assessment of oxidative stress at membrane level was measured by quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA deriving from the decomposition of poly-unsaturated fatty acids. The expression of Poly-ADP-Ribose-Polymerase (PARP, consequent to DNA fragmentation, was evidenced by immuno-histochemistry utilizing an antibody directed against an N-terminal fragment of the enzyme. Results The bioactivity of the drug was investigated on Hela cells. Cytoxicity was assessed by the Mosmann assay and by vital staining with trypan blue. The target of the molecule is most likely the cell membrane as shown by the significant increase of the intracellular concentration of malonyl-dihaldheyde. The increase of this compound, as a consequence of the treatment with PD166866, is suggestive of membrane lipoperoxidation. The TUNEL assay gave a qualitative, though clear, indication of DNA damage. Furthermore we demonstrate intracellular accumulation of poly-ADP-ribose polymerase I. This enzyme is a sensor of nicks on the DNA strands and this supports the idea that treatment with the drug induces cell

  14. False positive reaction due to endogenous biotin activity in glandular epithelium of decidua Reação falso positiva em epitélio glandular da decídua devido a atividade endógena de biotina

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    Liliana Cruz Spano

    2005-06-01

    Full Text Available Biotin-labeled probe was used in an in situ hybridisation assay to localize virus infection in formalin-fixed, paraffin embedded tissues taken from eleven abortion cases. Probes for human cytomegalovirus (HCMV, human Parvovirus B19 (B19 and human adenovirus type 2 (HAd2, were labeled with biotin-11-dUTP by nick-translation reaction. Streptavidin-alkaline-phosphatase (SAP was used to detect biotin, followed by 4-nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP solution. Positive reaction was observed in nucleus of glandular ephitelium cells of decidua either in positive or in negative control at first and second gestational trimester. The reaction was not inhibited with blocking solution for alkaline phosphatase endogenous activity and it persisted even with probes omission. The use of adequate negative control permitted to reveal the presence of nuclear biotin in glandular epithelium of decidua, responsible for false positivity in detection systems involving streptavidin biotin system (StrepABC. The stained cells resembled to cytophatic effect due to herpesvirus, which could induce further misinterpretation. The results obtained in this study strongly recommend that DNA detection by in situ hybridisation reaction in gestational endometrium should be done without using StrepABC system.Sondas marcadas com biotina foram utilizadas neste trabalho para detecção de infecção viral por hibridização in situ em tecidos fixados com formalina e embebidos em parafina de 11 casos obtidos de abortamento. Sondas para citomegalovírus humano (HCMV, parvovírus B19 humano (B19 e adenovírus humano tipo 2 (HAd2, foram marcadas com biotina-11-dUTP através da reação de nick-translation. Estreptavidina conjugada com fosfatase alcalina (SAP seguida por solução de 4-nitro-azul de tetrazolio/5-bromo-4-cloro-3-indolil fosfato (NBT/BCIP foram utilizadas para detecção da biotina após a reação de hibridização. Reação positiva foi

  15. Can Social Instability, Food Deprivation and Food Inequality Accelerate Neuronal Aging?

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    Shahnaz Mojarab

    2012-07-01

    Full Text Available Based on both animal and human studies, inequality in food intake and social instability has adverse effects on the health of individuals and the community. However, it is not known whether social instability, food deprivation and food inequality affect neuronal death and premature aging in young animals. To address this question, the effects of these adverse situations, histopathological changes in hippocampal pyramidal cells and aging process were investigated.Forty eight New Zeeland white male rabbits were divided into six groups and all of them were housed in similar conditions, with 2 animals per cage in a temperature-controlled colony room under light–dark cycle . All experimental animals were fed on standard rabbit commercial pellets and different social situations such as food deprivation, inequality in food intake, and unstable social status were applied to experimental groups during eight weeks. Afterward, lipofuscin accumulation and apoptosis, as main markers of aging, were compared to the control group by Long Ziehl Nelseen staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL reaction assay to reveal the rate of lipofuscin pigment accumulation and TUNEL-reactive apoptotic bodies in the hippocampal pyramidal cells. Serum cortisol level was also measured. Inequality in social situation raised chronic stress (i.e. food deprivation, social inequality and instability and caused significant changes in lipofuscin accumulation in hippocampal pyramidal cells in comparison to the control group (p<0.005 . The results also showed a significant increase in the ratio of apoptotic to normal cells in all of the stressed groups compared to the control group (p<0.05. Moreover, application of the social inequality and stresses alone or together modulated levels of cortisol in the experimental group. These findings suggest that food deprivation, inequality and social instability enhance the susceptibility of hippocampal

  16. Desvio da proporção de sexo e da integridade do DNA dos espermatozóides bovinos centrifugados em gradientes de densidade contínuos Alteration of sex ratio and DNA integrity of bovine sperm centrifuged in continuous density gradients

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    Alberto Lopes Gusmão

    2010-03-01

    Full Text Available O objetivo, neste trabalho, foi verificar o desvio da proporção de sexo e a presença de fragmentação do DNA, pela técnica de TUNEL (“In situ terminal deoxinucleotidyl transferase mediated dUTP nick end labeling assay”, em espermatozoides bovinos centrifugados em gradientes de densidade de Percoll ou OptiPrep durante a separação espermática. Doses de sêmen de touros foram descongeladas, e cerca de 40 milhões de espermatozoides foram depositados sobre cada gradiente de densidade compostos por Percoll ou OptiPrep com três camadas entre 1.110g/mL e 1.123g/mL, em tubos de 15mL, em que permaneceram por 24h a 4°C antes da deposição dos espermatozoides. Os tubos foram centrifugados a 500xg por 15min a 22°C. Os sobrenadantes foram aspirados, e os sedimentos, recuperados para verificação da fragmentação do DNA pela técnica de TUNEL. Obteve-se um desvio dos embriões produzidos in vitro para fêmeas no gradiente de Percoll (62% de fêmeas, em relação aos grupos OptiPrep e Controle (47,1 e 48,7% de fêmeas, respectivamente. Não foi detectada fragmentação do DNA dos espermatozoides nas amostras centrifugadas, tanto no gradiente de Percoll quanto de OptiPrep. Dessa forma, foi possível realizar a sexagem espermática, com uma maior porcentagem de espermatozoides X do que o grupo controle, por meio de metodologia mais simples e sem provocar danos ao DNA dos espermatozoides.The objective of the present study was to verify the sex ratio and presence of DNA fragmentation by TUNEL technique (In situ terminal deoxinucleotidyl transferase mediated dUTP nick end labeling assay in bovine spermatozoa centrifuged in density gradients of Percoll or OptiPrep during the sperm separation. Approximately 40 million of frozen/thawed bovine spermatozoa were deposited on each density gradient composed of Percoll or OptiPrep with three layers ranging from 1.110g/mL to 1.123g/mL in polystyrene tubes of 15mL. The tubes were kept at 4°C for 24h before

  17. Peran Kedelai (Glycine max L. dalam Pencegahan Apoptosis pada Cedera Jaringan Hati

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    Maya Tejasari

    2013-02-01

    Full Text Available Abstrak Pada liver injury akibat berbagai sebab, terjadi apoptosis sel yang sangat banyak yang dapat memengaruhi fungsi metabolik hati.  Isoflavon kedelai telah diketahui dapat mencegah apoptosis sel pada folikel ovarium dan osteoblas. Penelitian ini bertujuan untuk mengetahui pengaruh kedelai pada pencegahan apoptosis sel pada jaringan hati mencit yang diinduksi CCl4.  Penelitian dilakukan menggunakan 30 ekor mencit jantan galur DDY berumur 8─10 minggu yang dibagi dalam 6 kelompok perlakuan.  Kelompok 1 merupakan kontrol positif yang hanya diberi makanan pelet standar selama 3 minggu kemudian diberi 0,2 mL larutan CCl4 per oral selama 4 hari. Kelompok 2 merupakan kontrol negatif yang hanya diberi makanan pelet standar dan tidak diberi CCl4, sedangkan kelompok 3─6 merupakan kelompok uji yang selain diberi makanan pelet standar juga diberi kedelai dengan kadar berturut-turut 145,6 mg/hari, 218,4 mg/hari, 291,2 mg/hari dan 364 mg/hari selama 3 minggu kemudian diberi 0,2 mL larutan CCl4 peroral selama 4 hari.  Seluruh kelompok kemudian dikorbankan dan diambil organ hatinya untuk dilakukan pemeriksaan histokimia terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL.  Parameter yang diukur adalah jumlah apoptosis sel pada sayatan jaringan hati mencit menggunakan mikroskop cahaya.  Data disajikan dan dianalisis secara statistik menggunakan uji analysis of varians (ANOVA untuk menganalisis perbedaan antar kelompok. Hasil penelitian memperlihatkan bahwa dari hasil pemeriksaan imunohistokimia TUNEL tampak jumlah sel yang mengalami apoptosis pada kelompok yang diberi kedelai lebih sedikit dibandingkan dengan kelompok yang tidak diberi kedelai. Analisis uji ANOVA antara kelompok tersebut menunjukan perbedaaan yang signifikan dengan nilai p<0,05. Simpulan, bahwa pemberian kedelai dapat mencegah apoptosis sel pada jaringan hati mencit yang diinduksi CCl4. Kata kunci: Apoptosis, CCl4, isoflavon, kedelai, liver injury, TUNEL

  18. Modelo experimental de perfusão pulmonar ex vivo em ratos: avaliação histopatológica e de apoptose celular em pulmões preservados com solução de baixo potássio dextrana vs. solução histidina-triptofano-cetoglutarato An experimental rat model of ex vivo lung perfusion for the assessment of lungs regarding histopathological findings and apoptosis: low-potassium dextran vs. histidine-tryptophan-ketoglutarate

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    Edson Azevedo Simões

    2012-08-01

    Full Text Available OBJETIVO: Comparar os achados histopatológicos e de apoptose em pulmões de ratos preservados em soluções low-potassium dextran (LPD, baixo potássio dextrana, histidine-tryptophan-ketoglutarate (HTK, histidina-triptofano-cetoglutarato ou salina normal (SN em 6 h e 12 h de isquemia pela utilização de um modelo experimental de perfusão pulmonar ex vivo. MÉTODOS: Sessenta ratos Wistar foram anestesiados, randomizados e submetidos à perfusão anterógrada pela artéria pulmonar com uma das soluções preservadoras. Após a extração, os blocos cardiopulmonares foram preservados por 6 ou 12 h a 4ºC, sendo então reperfundidos com sangue homólogo em um sistema de perfusão ex vivo durante 60 min. Ao final da reperfusão, fragmentos do lobo médio foram extraídos e processados para histopatologia, sendo avaliados os seguintes parâmetros: congestão, edema alveolar, hemorragia alveolar, hemorragia, infiltrado inflamatório e infiltrado intersticial. O grau de apoptose foi avaliado pelo método TdT-mediated dUTP nick end labeling. RESULTADOS: A histopatologia demonstrou que todos os pulmões preservados com SN apresentaram edema alveolar após 12 h de isquemia. Não houve diferenças em relação ao grau de apoptose nos grupos estudados. CONCLUSÕES: No presente estudo, os achados histopatológicos e de apoptose foram semelhantes com o uso das soluções LPD e HTK, enquanto a presença de edema foi significativamente maior com o uso de SN.OBJECTIVE: To compare histopathological findings and the degree of apoptosis among rat lungs preserved with low-potassium dextran (LPD solution, histidine-tryptophan-ketoglutarate (HTK solution, or normal saline (NS at two ischemia periods (6 h and 12 h using an experimental rat model of ex vivo lung perfusion. METHODS: Sixty Wistar rats were anesthetized, randomized, and submitted to antegrade perfusion via pulmonary artery with one of the preservation solutions. Following en bloc extraction, the heart

  19. Chemopreventive response of diclofenac, a non-steroidal anti-inflammatory drug in experimental carcinogenesis Respuesta quimiopreventiva del diclofenaco, un fármaco antiinflamatorio no esteroideo en la carcinogénesis de colon experimental

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    M. Kaur Saini

    2009-12-01

    Full Text Available The chemopreventive response was evaluated of nonsteroidal anti-inflammatory drug, Diclofenac, a preferential cyclooxygenase-2 (COX-2 inhibitor in 1,2-dimethyhydrazine (DMH-induced colon cancer in rat model. The signs of neoplasm were evident in the animals receiving 30mg of DMH per kg body weight in a weekly s.c injection for six weeks. The putative biomarker of carcinogenesis was visible in the form of multiple plaque lesions in DMH treatment and then regression seen in those animals which also received an oral dose of Diclofenac, 8 mg/kg body weight whereas no such macroscopic neoplastic lesions were seen in the animals receiving Diclofenac only or the control animals receiving the vehicle of the drug. Histopathological results showed the presence of early aberrant changes in the form of severe dysplasia and also numerous crypt fissions in the apical surface of the colonic mucosa. A very high expression of COX-2 was seen in the colonic epithelium of DMH-treated rats, as analyzed by immunohistochemistry. Also, the apoptotic events were assessed by terminal deoxynucleotidyl dUTP nick end labeling (TUNEL assay, where the DMH group shows few number of TUNEL positive cells which dramatically increased in the Diclofenac treatment. The results suggest that Diclofenac could be an effective chemopreventive agent in colon cancer, where perhaps apoptosis plays a very dominant end effect in cancer cell killings.Se evaluó la respuesta quimiopreventiva del fármaco antiinflamatorio no esteroideo, diclofenaco, un inhibidor preferente de la ciclooxigenasa-2 (Cox-2, en el cáncer de colon inducido por 1,2-dimetilhidracina (DMH en un modelo de rata. Los signos de neoplasia fueron evidentes en los animales que recibieron 30 mg de DMH por kg de peso corporal mediante inyecciones s.c. semanales durante 6 semanas. El biomarcador putativo de la carcinogénesis fue visible en la forma de múltiples lesiones en placas con el tratamiento de DMH y la posterior regresi

  20. Mild maternal iron deficiency anemia induces DPOAE suppression and cochlear hair cell apoptosis by caspase activation in young guinea pigs.

    Science.gov (United States)

    Yu, Fei; Hao, Shuai; Zhao, Yue; Ren, Yahao; Yang, Jun; Sun, Xiance; Chen, Jie

    2014-01-01

    Iron deficiency (ID) anemia (IDA) alters auditory neural normal development in the mammalian cochlea. Previous results suggest that mild maternal IDA during pregnancy and lactation altered the hearing and nervous system development of the young offspring, but the mechanisms underlying the association are incompletely understood. The objective of this study was to evaluate the role of apoptosis in the development of sensory hair cells following mild maternal IDA during pregnancy and lactation. We established a maternal anemia model in female guinea pigs by using a mild iron deficient diet. The offspring were weaned on postnatal day (PND) 9 and then was given the iron sufficient diet. Maternal blood samples were collected on gestational day (GD) 21, GD 42, GD 63 and PND 9, serum level of iron (SI) or hemoglobin (Hb) was measured. Blood samples of pups were collected on PND 9 for SI measurement. On PND 24, pups were examined the distortion product otoacoustic emission (DPOAE) task, and then the cochleae were harvested for assessment of apoptosis by immunohistochemistry of cysteine-aspartic acid proteases 3/9 (caspase-3/9) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and by double immunofluorescence for the colocalization of TUNEL and caspase-3. Blood samples of pups were collected on PND 24 for SI and Hb measurements. Here we show that mild maternal IDA during pregnancy and lactation resulted in hearing impairment, decreased hair cell number, caspase-3/9 activation and increased apoptotic cell number of young guinea pigs. These results indicate a key role for apoptosis in inhibition of hair cell development, caused by mild maternal IDA during pregnancy and lactation.

  1. Regulating effects of arsenic trioxide on cell death pathways and inflammatory reactions of pancreatic acinar cells in rats

    Institute of Scientific and Technical Information of China (English)

    XUE Dong-bo; ZHANG Wei-hui; YUN Xiao-guang; SONG Chun; ZHENG Biao; SHI Xing-ye; WANG Hai-yang

    2007-01-01

    Background It is accepted that inflammatory cytokines play a key role in the development of acute pancreatitis, so blocking the initiation of inflammatory reactions may alleviate pathological changes of acute pancreatitis. We studied the regulatory effect of arsenic trioxide (As2O3) on apoptosis and oncosis of pancreatic acinar cells in vitro and in vivo and its therapeutic effect on acute pancreatitis.Methods Pancreatic acinar cells were isolated by collagenase digestion method. Apoptosis and oncosis of isolated pancreatic acinar cells were detected with Hoechst 33258+PI or Annexin V+PI double fluorescent staining. Amylase and lactate dehydrogenase release were measured. Acute pancreatitis was induced in Wistar rats by intraperitoneal injections of caerulein, and apoptosis was detected with terminal dUTP nick-end labeling method. Tumor necorsis factor α (TNF-α) mRNA, myeloperoxidase, nuclear factor-κB and histological grading of pancreatic damage were measured.Results There was an increased apoptosis but a decreased oncosis of pancreatic acinar cell after the treatment with As2O3. The levels of lactate dehydrogenase and amylase release were markedly decreased in As2O3 treated group.Myeloperoxidase content, TNF-α mRNA level, nuclear factor-κB activation and pathological score in As2O3 treated group were significantly lower than in the untreated group.Conclusions As2O3 can induce apoptosis and reduce oncosis of pancreatic acinar cell, thus resulting in reduced release of endocellular enzyme of acinar cells, reduced inflammatory cell infiltration and decreased the production of inflammatory cytokines, so that the outcome of alleviated pathological changes was finally achieved.

  2. Effects of 1, 25-Dihydroxyvitamin D3 on Experimental Autoimmune Myocarditis in Mice

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    Fen Hu

    2016-05-01

    Full Text Available Background/Aims: Myocarditis is an important inflammatory disease of the heart which causes life-threatening conditions. 1, 25(OH2 D3 has effects on multiple systems and diseases. The present study was aimed to investigate the effect of 1, 25(OH2 D3 on experimental autoimmune myocarditis (EAM, and explored the underlying mechanisms involved. Methods: EAM was induced by immunizing BALB/c mice with cardiac α-myosin heavy chain peptides (MyHC-α. 1, 25(OH2 D3 (1,000 ng/kg once or vehicle was administered intraperitoneally every other day during the entire experiment. On day 21, transthoracic echocardiography was performed and cardiac inflammatory infiltration was detected by hematoxylin and eosin (HE. The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL assay, and Western blots for the expression of protein caspase-3 and cleaved-caspase3 were used to evaluate apoptosis. Transmission electron microscopy and Western blots for the expression of protein Beclin-1, LC3B, and P62 were used to evaluate autophagy. Results: The ratio of heart weight/body weight was significantly reduced in 1, 25(OH2 D3 -treated EAM mice, compared with vehicle -treated ones. 1, 25(OH2 D3 treatment improved cardiac function, diminished cell infiltration in cardiac, suppressed myocardial apoptosis, decreased the number of autophagosomes, and decreased the protein expression of Beclin-1, LC3-II and p62. Conclusions: The present results demonstrated that administration of 1, 25(OH2 D3 decreased EAM severity. 1, 25(OH2 D3 treatment may be a feasible therapeutic approach for EAM.

  3. Optimization of time for neural stem cells transplantation for brain stroke in rats.

    Science.gov (United States)

    Ziaee, Seyyed Mohyeddin; Tabeshmehr, Parisa; Haider, Khawaja Husnain; Farrokhi, Majidreza; Shariat, Abdolhamid; Amiri, Atena; Hosseini, Seyed Mojtaba

    2017-01-01

    Despite encouraging data in terms of neurological outcome, stem cell based therapy for ischemic stroke in experimental models and human patients is still hampered by multiple as yet un-optimized variables, i.e., time of intervention, that significantly influence the prognosis. The aim of the present study was to delineate the optimum time for neural stem cells (NSCs) transplantation after ischemic stroke. The NSCs were isolated from 14 days embryo rat ganglion eminence and were cultured in NSA medium (neurobasal medium, 2% B27, 1% N2, bFGF 10 ng/mL, EGF 20 ng/mL and 1% pen/strep). The cells were characterized for tri-lineage differentiation by immunocytochemistry for tubulin-III, Olig2 and GFAP expression for neurons, oligodendrocytes and astrocyte respectively. The NSCs at passage 3 were injected intraventricularly in a rodent model of middle-cerebral artery occlusion (MCAO) on stipulated time points of 1 & 12 h, and 1, 3, 5 and 7 days after ischemic stroke. The animals were euthanized on day 28 after their respective treatment. dUTP nick end labeling (TUNEL) assay and Caspase assay showed significantly reduced number of apoptotic cells on day 3 treated animals as compared to the other treatment groups of animals. The neurological outcome showed that the group which received NSCs 3 days after brain ischemia had the best neurological performance. The optimum time for NSCs transplantation was day 3 after ischemic stroke in terms of attenuation of ischemic zone expansion and better preserved neurological performance.

  4. Propofol Ameliorates Calpain-induced Collapsin Response Mediator Protein-2 Proteolysis in Traumatic Brain Injury in Rats

    Science.gov (United States)

    Yu, Yun; Jian, Min-Yu; Wang, Yun-Zhen; Han, Ru-Quan

    2015-01-01

    Background: Collapsin response mediator protein-2 (CRMP2), a multifunctional cytosolic protein highly expressed in the brain, is degraded by calpain following traumatic brain injury (TBI), possibly inhibiting posttraumatic neurite regeneration. Lipid peroxidation (LP) is involved in triggering postinjury CRMP2 proteolysis. We examined the hypothesis that propofol could attenuate LP, calpain-induced CRMP2 degradation, and brain injury after TBI. Methods: A unilateral moderate controlled cortical impact injury was induced in adult male Sprague-Dawley rats. The animals were randomly divided into seven groups: Sham control group, TBI group, TBI + propofol groups (including propofol 1 h, 2 h, and 4 h groups), TBI + U83836E group and TBI + fat emulsion group. The LP inhibitor U83836E was used as a control to identify that antioxidation partially accounts for the potential neuroprotective effects of propofol. The solvent of propofol, fat emulsion, was used as the vehicle control. Ipsilateral cortex tissues were harvested at 24 h post-TBI. Immunofluorescent staining, Western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling were used to evaluate LP, calpain activity, CRMP2 proteolysis and programmed cell death. The data were statistically analyzed using one-way analysis of variance and a paired t-test. Results: Propofol and U83836E significantly ameliorated the CRMP2 proteolysis. In addition, both propofol and U83836E significantly decreased the ratio of 145-kDa αII-spectrin breakdown products to intact 270-kDa spectrin, the 4-hydroxynonenal expression and programmed cell death in the pericontusional cortex at 24 h after TBI. There was no difference between the TBI group and the fat emulsion group. Conclusions: These results demonstrate that propofol postconditioning alleviates calpain-mediated CRMP2 proteolysis and provides neuroprotective effects following moderate TBI potentially by counteracting LP and reducing calpain activation

  5. Over-starvation aggravates intestinal injury and promotes bacterial and endotoxin translocation under high-altitude hypoxic environment

    Institute of Scientific and Technical Information of China (English)

    Qi-Quan Zhou; Ding-Zhou Yang; Yong-Jun Luo; Su-Zhi Li; Fu-Yu Liu; Guan-Song Wang

    2011-01-01

    AIM: To study whether over-starvation aggravates intestinal mucosal injury and promotes bacterial and endotoxin translocation in a high-altitude hypoxic environment. METHODS: Sprague-Dawley rats were exposed to hypobaric hypoxia at a simulated altitude of 7000 m for 72 h. Lanthanum nitrate was used as a tracer to detect intestinal injury. Epithelial apoptosis was observed with terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Serum levels of diamino oxidase (DAO), malondialdehyde (MDA), glutamine (Gln), superoxide dismutase (SOD) and endotoxin were measured in intestinal mucosa. Bacterial translocation was detected in blood culture and intestinal homogenates. In addition, rats were given Gln intragastrically to observe its protective effect on intestinal injury. RESULTS: Apoptotic epithelial cells, exfoliated villi and inflammatory cells in intestine were increased with edema in the lamina propria accompanying effusion of red blood cells. Lanthanum particles were found in the intercellular space and intracellular compartment. Bacterial translocation to mesenteric lymph nodes (MLN) and spleen was evident. The serum endotoxin, DAO and MDA levels were significantly higher while the serum SOD, DAO and Gln levels were lower in intestine (P < 0.05). The bacterial translocation number was lower in the high altitude hypoxic group than in the high altitude starvation group (0.47 ± 0.83 vs 2.38 ± 1.45, P < 0.05). The bacterial translocation was found in each organ, especially in MLN and spleen but not in peripheral blood. The bacterial and endotoxin translocations were both markedly improved in rats after treatment with Gln. CONCLUSION: High-altitude hypoxia and starvation cause severe intestinal mucosal injury and increase bacterial and endotoxin translocation, which can be treated with Gln.

  6. The effects of heavy ion particles on the developing murine cerebellum, with special reference to cell death

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Chikako; Yaoi, Takeshi; Fushiki, Shinji [Kyoto Prefectural Univ. of Medicine (Japan). Research Inst. for Neurological Diseases and Geriatrics; Nojima, Kumie [National Inst. of Radiological Sciences, Chiba (Japan). Internatinal Space Radiation Lab.

    2003-07-01

    We report here the effects of heavy ion beams on postnatal mouse cerebellar development, with reference to cell death. Eight-day-old B6C3F1 mice were irradiated with single doses of 0.1, 0.25, 0.5, 1.0, and 2.0 Gy, using a carbon beam of 290 MeV delivered from a heavy ion medical accelerator in Chiba (HIMAC). To compare the effects of X-rays with those of accelerated carbon ions, 8-day-old mice were exposed to X-rays single doses of 0.1, 0.25, 0.5, 1.0, and 2.0 Gy, respectively. Pups were fixed at 1, 6, 12 and 24 hr after exposure to HIMAC beams or X-rays. Four-{mu}m-thick parasagittal sections of the cerebella were processed for hematoxylin-eosin staining as well as for staining with the TUNEL (terminal dUTP nick-end labeling) technique. The density of fragmented nuclei in the external granular layer increased with time, peaking at 6 hr after exposure, in both the HIMAC and X-irradiated groups. In the HIMAC groups, the density was significantly higher in those animals exposed to 0.25 Gy or more compared to 0 Gy, whereas in the X-irradiated groups it was significantly higher in those mice exposed to 0.5 Gy or more. Electron microscopic examinations revealed chromatin condensation in the cell nuclei in the HIMAC groups. This is the first in vivo evidence that apoptotic cell death is induced in developing mouse cerebellum after exposure to heavy ion particles. The difference in the frequency of dying cells between exposure to heavy ion particles and to X-rays may reflect the high linear energy transfer (LET) associated with a heavy ion beam. (author)

  7. Protective role of adiponectin in a rat model of intestinal ischemia reperfusion injury

    Science.gov (United States)

    Liu, Xu-Hui; Yang, Yue-Wu; Dai, Hai-Tao; Cai, Song-Wang; Chen, Rui-Han; Ye, Zhi-Qiang

    2015-01-01

    AIM: To determine the potential protective role of adiponectin in intestinal ischemia reperfusion (I/R) injury. METHODS: A rat model of intestinal I/R injury was established. The serum level of adiponectin in rats with intestinal I/R injury was determined by enzyme-linked immunosorbent assay (ELISA). The serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were also measured by ELISA. Apoptosis of intestinal cells was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The production of malondialdehyde (MDA) and superoxide dismutase (SOD) and villous injury scores were also measured. RESULTS: Adiponectin was downregulated in the serum of rats with intestinal I/R injury compared with sham rats. No significant changes in the expression of adiponectin receptor 1 and adiponectin receptor 2 were found between sham and I/R rats. Pre-treatment with recombinant adiponectin attenuated intestinal I/R injury. The production of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, in rats with intestinal I/R injury was reduced by adiponectin pre-treatment. The production of MDA was inhibited, and the release of SOD was restored by adiponectin pre-treatment in rats with intestinal I/R injury. Adiponectin pre-treatment also inhibited cell apoptosis in these rats. Treatment with the AMP-activated protein kinase (AMPK) signaling pathway inhibitor, compound C, or the heme oxygenase 1 (HO-1) inhibitor, Snpp, attenuated the protective effects of adiponectin against intestinal I/R injury. CONCLUSION: Adiponectin exhibits protective effects against intestinal I/R injury, which may involve the AMPK/HO-1 pathway. PMID:26715807

  8. Effect of genistein on mouse blastocyst development in vitro

    Institute of Scientific and Technical Information of China (English)

    Wen-hsiung CHAN; Hsiang-yu LU; Nion-heng SHIAO

    2007-01-01

    Aim: To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro. Methods: Mouse blastocysts were incubated in medium with or without genistein (25 or 50 μmol/L) or daidzein (50 μmol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis. Results: TUNEL staining and Annexin-V/PI staining. showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment de-creased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis. Conclusions: Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein

  9. Evaluation of Effects of Quercetin (3, 3’, 4’, 5, 7-pentohidroxyflavon on Apoptosis and Telomerase Enzyme Activity in MCF-7 and NIH-3T3 Cell Lines Compared with Tamoxifen

    Directory of Open Access Journals (Sweden)

    Ayşe Ak

    2011-09-01

    Full Text Available Objective: Quercetin has been shown to inhibit the proliferation of cancer cells. Tamoxifen is used for breast cancer. In this study, we have aimed to investigate the effects of quercetin and tamoxifen on telomerase enzyme activity and apoptosis in two cell lines.Methods: In this study, 10, 50 and 100 µM of quercetin and tamoxifen were used to treat MCF-7 and NIH-3T3. Apoptosis was determined by the TUNEL method (Terminal Deoxynucleotide Transferase dUTP Nick End Label and telomerase enzyme activity was determined by ELISA (Enzyme-Linked Immunosorbent Assay. Results: In the NIH3T3 cell line, only 100 µM quercetin and tamoxifen induced significant apoptosis. In the MCF-7 cell line 10 µM and 100 µM quercetin in the 24th hour and 100 µM quercetin in the 72nd hour induce apoptosis. In 24th and 48th hours, 50 µM tamoxifen and 100 µM tamoxifen in the 24th and 48th hours induce apoptosis in the MCF-7 cell line. In MCF-7 and NIH-3T3 cell lines, all doses of quercetin and tamoxifen reduced telomerase enzyme activity compared to the control group. Conclusion: In this study, it was shown that quercetin has similar effects to tamoxifen. However quercetin induces apoptosis more than decreasing telomerase enzyme activities, being different from tamoxifen. We hope that the findings will assist in developing new therapeutic pathways for preventing breast cancer. However, there should be many more studies in order to discover quercetin and other potential drugs.

  10. Apigenin suppresses the growth of colorectal cancer xenografts via phosphorylation and up-regulated FADD expression.

    Science.gov (United States)

    Wang, Qi Rui; Yao, Xue Qing; Wen, Ge; Fan, Qin; Li, Ying-Jia; Fu, Xiu Qiong; Li, Chang Ke; Sun, Xue Gang

    2011-01-01

    Apigenin is a flavonoid belonging to the flavone structural class. It has been implicated as a chemopreventive agent against prostate and breast cancers. However, to the best of our knowledge, no published data are available regarding apigenin in colorectal cancer (CRC). The effects and mechanisms of apigenin on CRC may vary significantly. This study aimed to analyze the effects of apigenin on the growth of CRC xenografts in nude mice derived from SW480, as well as to investigate the underlying mechanisms. Whole-body fluorescence imaging is an inexpensive optical system used to visualize gene expression in small mammals using reporter genes, such as eGFP as a reporter. In our study, the expression of eGFP may reflect the size of the tumor. A terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay showed that apigenin promoted the apoptosis of CRC cells. Furthermore, the expression of five genes related to the proliferation and apoptosis of CRC, i.e., cyclin D1, BAG-1, Bcl-2, yrdC and Fas-associated protein with death domain (FADD), was detected by real-time quantitative RT-PCR. Among these genes, the up-regulated expression of FADD was noted in CRC xenograft tumors treated with apigenin. Immunohistochemistry and Western blotting confirmed the results at the protein level. Furthermore, Western blot analysis showed that apigenin induced the phosphorylation of FADD. Our findings suggest that apigenin enhances the expression of FADD and induces its phosphorylation, which may cause apoptosis of CRC cells and inhibition of tumor growth.

  11. Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

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    Shadab, Md.; Jha, Baijayanti; Asad, Mohammad; Deepthi, Makaraju; Kamran, Mohd.; Ali, Nahid

    2017-01-01

    Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. Conclusions KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function. PMID:28170432

  12. Conditional Knockout of Src Homology 2 Domain-containing Protein Tyrosine Phosphatase-2 in Myeloid Cells Attenuates Renal Fibrosis after Unilateral Ureter Obstruction

    Institute of Scientific and Technical Information of China (English)

    Jing-Fei Teng; Kai Wang; Yao Li; Fa-Jun Qu; Qing Yuan; Xin-Gang Cui; Quan-Xing Wang

    2015-01-01

    Background:Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase.Studies have revealed its roles in various disease,however,whether SHP-2 involves in renal fibrosis remains unclear.The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.Methods:Myeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system,and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO).The total collagen deposition in the renal interstitium was assessed using picrosirius red stain.F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium.Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney.Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.Results:Src homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO.Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice.Meanwhile,the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice.However,no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.Conclusions:Our observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis,and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

  13. The anti-tumor effect of p53 gene-loaded hydroxyapatite nanoparticles in vitro and in vivo

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    Zhao, Ruibo; Yang, Xinyan; Chen, Cen; Chen, Kan; Wang, Shibing; Xie, Chungang; Ren, Xiaoyuan; Kong, Xiangdong

    2014-04-01

    This research focused on anti-tumor effect of pEGFP-C1-p53 (p53) gene-loaded hydroxyapatite (HAp) nanoparticles in vitro and in vivo. Four kinds of HAp nanoparticles, spherical HAp nanoparticles (S-HAp, diameter: 50 nm), needle-like HAp nanoparticles (N-HAp, average length: 110 nm and width: 30 nm), rod-like HAp nanoparticles (R-HAp, average length: 100 nm and width: 30 nm), and short-rod-like HAp nanoparticles (SR-HAp, average length: 40 nm and width: 30 nm), were prepared initially. The HAp nanoparticles with or without being modified by PEI (named HAp and HAp-PEI, respectively) have excellent biocompatibility as shown by MTT assay and crystal violet staining tests. Then, the subsequent MTT, Hocehst staining tests, and Western blot showed that the killing effect of p53-loaded HAp-PEI (HAp-PEI-p53) was effective with fair selectivity toward Hep-3B and HuH-7 cells' cell lines. Moreover, HAp-PEI-p53 could inhibit the tumor growth in vivo, and the mechanism of tumor growth inhibition was verified by the hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, P53 protein immunohistochemistry, and transmission electron microscope of the tumor cell in vivo. We found that HAp-PEI-p53 has good anti-cancer effect in vitro and in vivo, especially for the S-HAp-PEI-p53. Tumor metastasis could be suppressed significantly by the S-HAp-PEI-p53 and N-HAp-PEI-p53 treatments by the in vivo imaging system. All these results lead to the conclusion that the particle sizes of HAp ranging from 100 to 200 nm are appropriate for cancer gene therapy and may be widely used in anti-cancer investigation.

  14. Inhibitory effects of omega-3 fatty acids on early brain injury after subarachnoid hemorrhage in rats: Possible involvement of G protein-coupled receptor 120/β-arrestin2/TGF-β activated kinase-1 binding protein-1 signaling pathway.

    Science.gov (United States)

    Yin, Jia; Li, Haiying; Meng, Chengjie; Chen, Dongdong; Chen, Zhouqing; Wang, Yibin; Wang, Zhong; Chen, Gang

    2016-06-01

    Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected by mild cognitive impairment, and mediate potent anti-inflammatory via G protein-coupled receptor 120 (GPR120) signal pathway. Neuron dysfunction and inflammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids was administered by oral gavage at 1g/kg body weight once every 24h. Specific siRNA for GPR120 was exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, fluoro-Jade B staining, and neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed that omega-3 fatty acids effectively suppressed SAH-induced elevation of inflammatory factors, including cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition, omega-3 fatty acids could inhibit phosphorylation of transforming growth factor β activated kinase-1 (TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B through regulating GPR120/β-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inflammatory effects via β-arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids as part of a daily diet may reduce EBI in an experimental rat model of SAH.

  15. A Combination of Podophyllotoxin and Rutin Alleviates Radiation-Induced Pneumonitis and Fibrosis through Modulation of Lung Inflammation in Mice

    Science.gov (United States)

    Verma, Savita; Kalita, Bhargab; Bajaj, Sania; Prakash, Hridayesh; Singh, Ajay Kumar; Gupta, Manju Lata

    2017-01-01

    Pneumonitis and pulmonary fibrosis are predominant consequences of radiation exposure, whether planned or accidental. The present study, demonstrates radioprotective potential of a formulation, prepared by combining podophyllotoxin and rutin (G-003M), in mice exposed to 11 Gy thoracic gamma radiation (TGR). Treated mice were observed for survival and other symptomatic features. Formation of reactive oxygen species (ROS)/nitric oxide (NO) was measured in bronchoalveolar lavage cells. DNA damage and cell death were assessed in alveolar cells by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Total protein (TP), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were measured in bronchoalveolar lavage fluid (BALF)/serum of mice to assess lung vascular permeability. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), cluster of differentiation 45, inducible nitric oxide synthase (iNOS), and nitrotyrosine were also estimated in lungs/BALF of differentially treated mice. Our observations revealed 100% survival in G-003M-pretreated mice against 66.50% in 11 Gy TGR exposed. Other symptoms like reduction in graying of hair, weight loss, and breathing rate were also observed in pretreated groups. Significant decline in ROS/NO and cell death in formulation pretreated mice were also observed. Decreased level of TP, LDH, and ALP in BALF/serum samples revealed G-003M-induced inhibition in lung permeability. Level of IL-6, TNF-α, and TGF-β1 in the lungs of these mice was found corresponding to control group at 8 weeks posttreatment. On the contrary, these cytokines raised significantly in 11 Gy TGR-exposed mice. Lung pneumonitis and fibrosis were found significantly countered in these mice. The observations revealed that G-003M could regulate immune system by curtailing radiation-induced oxidative and inflammatory stress, which has helped in minimizing radiation-inflicted pneumonitis and

  16. 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.

  17. Acute exposure to vibration is an apoptosis-inducing stimulus in the vocal fold epithelium.

    Science.gov (United States)

    Novaleski, Carolyn K; Kimball, Emily E; Mizuta, Masanobu; Rousseau, Bernard

    2016-10-01

    Clinical voice disorders pose significant communication-related challenges to patients. The purpose of this study was to quantify the rate of apoptosis and tumor necrosis factor-alpha (TNF-α) signaling in vocal fold epithelial cells in response to increasing time-doses and cycle-doses of vibration. 20 New Zealand white breeder rabbits were randomized to three groups of time-doses of vibration exposure (30, 60, 120min) or a control group (120min of vocal fold adduction and abduction). Estimated cycle-doses of vocal fold vibration were extrapolated based on mean fundamental frequency. Laryngeal tissue specimens were evaluated for apoptosis and gene transcript and protein levels of TNF-α. Results revealed that terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was significantly higher after 120min of vibration compared to the control. Transmission electron microscopy (TEM) revealed no significant effect of time-dose on the mean area of epithelial cell nuclei. Extrapolated cycle-doses of vibration exposure were closely related to experimental time-dose conditions, although no significant correlations were observed with TUNEL staining or mean area of epithelial cell nuclei. TUNEL staining was positively correlated with TNF-α protein expression. Our findings suggest that apoptosis can be induced in the vocal fold epithelium after 120min of modal intensity phonation. In contrast, shorter durations of vibration exposure do not result in apoptosis signaling. However, morphological features of apoptosis are not observed using TEM. Future studies are necessary to examine the contribution of abnormal apoptosis to vocal fold diseases.

  18. Relevance of LIF and EGF on Mouse Preimplantation Embryo Development

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    Iraj Amiri

    2008-01-01

    Full Text Available Objective: Recent evidence suggests that Leukemia Inhibitory Factor (LIF, a member ofinterleukin-6 family, has biological actions on preimplantation embryo development. Alsoit is established that Epidermal Growth Factor (EGF, a strong mitosis-promoting agent,improves the preimplantation embryo development by increasing the cell metabolism andproliferation. The purpose of the present study is to investigate the effects of these factors,alone and in combination together, on preimplantation and development of the embryo.Materials and Methods: Six to eight weeks old NMRI mice were super ovulated by injectionof 10IU PMSG and 10IU hCG, then the mated mice were killed 46 hours later. Theiroviducts were flushed, two-cell embryos collected and divided randomly to the four groupsas following: Control, treatment 1 (LIF, treatment 2 (EGF, treatment 3 (LIF+EGF. In eachgroup, the embryos were cultured in an incubator at 37°C with 5% CO2 and 90% humidityfor 72hrs. The state of embryo development was evaluated in 24,36,48,60 and 72hrsfollowing the embryos cultures. By the end of the cultures, cell apoptosis was studiedby the terminal deoxynucleotidyl transferas-mediated dUTP nick end-labeling (TUNELtechnique.Results: Significant difference was detected in the rate of hatching in the LIF and LIF+EGFgroups. This difference was also seen in the rate of blastocyst formation after 36hrs(p<0.05 and in the average of the total cell number (p<0.05 after 72hrs. In comparison tothe apoptotic index, there was no significant difference between the control and treatmentgroups.Conclusion: The findings in this study show a beneficial effect of LIF and EGF on theblastocyst formation, hatching and its total cell numbers in vitro.

  19. Studies on the correlation with olfactory dysfunction in a transgenic mice model of Alzheimer's disease

    Science.gov (United States)

    Rasheed, Ameer; Lee, Ji Hye; Suh, Yoo-Hun; Moon, Cheil

    2013-05-01

    Alzheimer's disease (AD) is a progressively debilitating neurodegenerative disorder characterized by the presence of proteinaceous deposits in the brain. AD often results in olfactory dysfunction and impaired olfactory perceptual acuity may be a potential biomarker for early diagnosis of AD. Until recently, there is no Alzheimer's nanoscope or any other high-end microscope developed to be capable of seeing buried feature of AD clearly. Modern neuroimaging techniques are more effective only after the occurrence of cognitive impairment. Therefore, early detection of Alzheimer's disease is critical in developing effective treatment of AD. H and E (Haematoxyline and Eosin) staining is performed for examining gross morphological changes, while TUNEL (transferase (TdT)-mediated dUTP nick end labeling) staining for monitoring neuronal death in the olfactory epithelium (OE). Furthermore, immunohistochemistry and western blot are performed to examine β-amyloid protein expression. AD model animals were Tg2576 (transgenic mice that overexpress a mutated form of the Aβ precursor protein), and 6 month (before onset of AD symptoms) and 14 month (after onset of AD symptoms) old WT (wild type) and transgenic mice were compared in their olfactory system. We found that in OE of Tg2576 mice, thickness and total number of cells were decreased, while the numbers of TUNEL-positive neurons, caspase-3 activation were significantly increased compared with age-matched WT. Our results demonstrate that the olfactory system may get deteriorated before onset of AD symptoms. Our findings imply that an olfactory biopsy could be served as an early and relatively simple diagnostic tool for potential AD patients.

  20. Effects of salmeterol on skeletal muscle cells: metabolic and proapoptotic features.

    Science.gov (United States)

    Duranti, Guglielmo; La Rosa, Piergiorgio; Dimauro, Ivan; Wannenes, Francesca; Bonini, Sergio; Sabatini, Stefania; Parisi, Paolo; Caporossi, Daniela

    2011-12-01

    Salmeterol is a β2-adrenergic receptor agonist widely used for the treatment of asthma and chronic obstructive pulmonary disease. It has been shown that salmeterol is also used at supratherapeutic doses as performance-enhancing substance in sport practice. Although the abuse of β-agonists might determine some adverse effects, the molecular effects of salmeterol on skeletal muscle cells remain unclear. We evaluated the effects of salmeterol (0.1-10 μM) on both proliferative and differentiated rat L6C5 and mouse C2C12 skeletal muscle cell lines. The metabolic effects were evaluated by glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase, citrate synthase, 3-OH acyl-CoA dehydrogenase, and alanine transglutaminase activities. Cytotoxic and apoptotic effects were analyzed by 3-(4,5-dimethylthiazol-1)-5-(3-carboxymeth-oxyphenyl)-2H-tetrazolium, trypan blue exclusion assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Western blot analysis, and immunofluorescence staining. We showed that salmeterol reduced the growth rate of proliferating cells in a dose- and time-dependent manner (6-48 h). An increase in oxidative metabolism was found after 6 h in C2C12 and L6C5 myoblasts and in C2C12 myotubes with respect to control cells, while in L6C5 myotubes, anaerobic metabolism prevailed. Exposure of myoblasts and myotubes for 48 and 72 h at high salmeterol concentrations induced apoptosis by the activation of the intrinsic apoptotic pathway, as confirmed by the modulation of the apoptotic proteins Bcl-xL, caspase-9, and poly (ADP-ribose) polymerase and by the cytoplasmic release of Smac/DIABLO. Altogether, our results demonstrate that short-term supratherapeutic salmeterol exposure increased oxidative metabolic pathways on skeletal muscle cells, whereas prolonged treatment inhibits cell growth and exerts either a cytostatic or a proapoptotic effect in a time- and dose-dependent way.

  1. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke.

  2. Expression of fragile histidine triad in primary hepatocellular carcinoma and its relation with cell proliferation and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ke-Jun Nan; Zhi-Ping Ruan; Zhao Jing; Hai-Xia Qin; Hong-Yan Wang; Hui Guo; Rui Xu

    2005-01-01

    AIM: To evaluate the expression of fragile histidine triad (FHIT) gene protein, product of a candidate tumor suppressor,and to investigate the relationship between FHIT, cell apoptosis and proliferation, and pathological features of primary hepatocellular carcinoma (HCC).METHODS: Forty-seven HCC and ten normal liver specimens were collected during surgical operation between 2001and 2003. FHIT and proliferating cell nuclear antigen (PCNA)expression were detected by immunohistochemistry, and apoptotic level was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay on the tissue sections.RESULTS: All normal liver tissues showed a strong expression of FHIT, whereas 28 of 47 (59.6%) carcinomas showed a significant loss or absence of FHIT expression (P = 0.001).The proportion of reduced FHIT expression in those carcinomas at stages Ⅲ-Ⅳ (70.6%) and in those with extrahepatic metastasis (86.7%) showed an increasing trend compared with those at stages Ⅰ-Ⅱ (30.8%, P= 0.013) and those without metastasis (46.9%, P = 0.010) respectively. Apoptotic incidence in advanced TNM stage carcinoma and those with positive FHIT expression was higher than that in early stage carcinoma (P = 0.030) and in those with negative FHIT expression (P = 0.044) respectively. The proliferating potential of hepatocellular carcinoma was associated with FHIT expression (P = 0.016) and the aggressive feature (P = 0.019). Kaplan-Meier analysis demonstrated that the survival time of these 47 patients correlated with TNM stage,FHIT expression and metastasis.CONCLUSION: There is marked loss or absence of FHIT expression, as well as abnormal apoptosis-proliferation balance in HCC. FHIT may play an important role in carcinogenesis and development of HCC.

  3. Anti-tumour activities of fucoidan from the aquatic plant Utricularia aurea lour

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    Wilaiwan Chotigeat

    2005-12-01

    Full Text Available Fucoidan, a sulfated polysaccharide with several biological activities, is usually isolated from marine seaweeds or from echinoderms. Here, we report on the anti-tumour activity of fucoidan isolated from the aquatic plant Utricularia aurea Lour (Lentibulariaceae. A crude extract (CE prepared by incubating U.aurea with hot water at 95ºC for 12 hr was partially purified by Sephadex G-50, eluting with a 50mM sodium acetate buffer, at pH 5.0, containing 0.5M NaCl. Partially purified fucoidan (PPF had a 3- fold increase in fucose content when compared with the CE and a molecular weigÄt of 11.6 kDa as determined by Sephadex G-200. Chemical analysis showed that CE consisted of 62.5% glucuronic acid, 5.0% fucose, 1.7% sulfate and 12.0% proteins while PPF consisted of 65.0% glucuronic acid, 15.3% fucose, 2.1 % sulfate and 8.3% proteins.The anti-tumour activity of the CE and PPF was determined by the MTT test. The CE at 125 μg/mL fucoidan and PPF at 250 μg/mL inhibited the growth of KB cells (a nasopharynx tumour cell line, but did not inhibit that of normal fibroblast cells. The inhibition was postulated to occur via apoptosis as significantlymore apoptotic cells were found after treatment than in the untreated KB cells (P<0.05 by the TUNNEL (TdT-mediated dUTP Nick-End Labelling assay.

  4. The effects of exposure to electromagnetic field on rat myocardium.

    Science.gov (United States)

    Kiray, Amac; Tayefi, Hamid; Kiray, Muge; Bagriyanik, Husnu Alper; Pekcetin, Cetin; Ergur, Bekir Ugur; Ozogul, Candan

    2013-06-01

    Exposure to electromagnetic fields (EMFs) causes increased adverse effects on biological systems. The aim of this study was to investigate the effects of EMF on heart tissue by biochemical and histomorphological evaluations in EMF-exposed adult rats. In this study, 28 male Wistar rats weighing 200-250 g were used. The rats were divided into two groups: sham group (n = 14) and EMF group (n = 14). Rats in sham group were exposed to same conditions as the EMF group except the exposure to EMF. Rats in EMF group were exposed to a 50-Hz EMF of 3 mT for 4 h/day and 7 days/week for 2 months. After 2 months of exposure, rats were killed; the hearts were excised and evaluated. Determination of oxidative stress parameters was performed spectrophotometrically. To detect apoptotic cells, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 immunohistochemistry were performed. In EMF-exposed group, levels of lipid peroxidation significantly increased and activities of superoxide dismutase and glutathione peroxidase decreased compared with sham group. The number of TUNEL-positive cells and caspase-3 immunoreactivity increased in EMF-exposed rats compared with sham. Under electron microscopy, there were mitochondrial degeneration, reduction in myofibrils, dilated sarcoplasmic reticulum and perinuclear vacuolization in EMF-exposed rats. In conclusion, the results show that the exposure to EMF causes oxidative stress, apoptosis and morphologic damage in myocardium of adult rats. The results of our study indicate that EMF-related changes in rat myocardium could be the result of increased oxidative stress. Further studies are needed to demonstrate whether the exposure to EMF can induce adverse effects on myocardium.

  5. Studies on arsenic trioxide induced mice melanoma Cloundman S91 cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ruzhi Zhang; Wenyuan Zhu; Chen Wang

    2005-01-01

    Objective: To invesfgate the effects of various concentrations As2O3 on malignant melanoma. Methods: The viability of Cloundman melanoma S91 cells treated with As2O3 was measured by MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] assay. The apoptosis was determined by transferase-mediated dUTP nick end labeling (TUNEL) detection, and the morphology of apoptotic cells was observed through transmission electron microscopy (TEM). The cells growth phase were analyzed by flow cytometry (FCM). Results: A time-and dose-dependent decrease in cell viability was induced in S91 cells after treatment with As2Os at the concentration of 1-5 μmol/L respectively. The TUNEL indices were 0.033 ± 0.018, 0.062 ± 0.012, 0.102 ± 0.016, 0.132 ± 0.031,and 0.162 ± 0.027 respectively, which were much higher compared with the control group (0.017 ± 0.004, P < 0.01). The flow cytometry showed that hypodiploid peak after treatment with 3 μmol/L and 5 μmol/L of As2O3 for 48 h were 9.99% and 17.59% respectively,which increased significantly compared with the control cells (3.05% P < 0.01, ). The apoptotic morphology observed by transmission electron microscope showed the chromatin became condensed and attached to the inner surface of nuclear membrane. Conclusion: Arsenic trioxide can induce melanoma Cloundman S91 cell apoptosis at the concentration of 1-5 μmol/L, which will provide enhanced benefit in melanoma therapy.

  6. Neuroprotective Effect of Scutellarin on Ischemic Cerebral Injury by Down-Regulating the Expression of Angiotensin-Converting Enzyme and AT1 Receptor.

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    Wenjuan Wang

    Full Text Available Previous studies have demonstrated that angiotensin-converting enzyme (ACE is involved in brain ischemic injury. In the present study, we investigated whether Scutellarin (Scu exerts neuroprotective effects by down-regulating the Expression of Angiotensin-Converting Enzyme and AT1 receptor in a rat model of permanent focal cerebral ischemia.Adult Sprague-Dawley rats were administrated with different dosages of Scu by oral gavage for 7 days and underwent permanent middle cerebral artery occlusion (pMCAO. Blood pressure was measured 7 days after Scu administration and 24 h after pMCAO surgery by using a noninvasive tail cuff method. Cerebral blood flow (CBF was determined by Laser Doppler perfusion monitor and the neuronal dysfunction was evaluated by analysis of neurological deficits before being sacrificed at 24 h after pMCAO. Histopathological change, cell apoptosis and infarct area were respectively determined by hematoxylin-eosin staining, terminal deoxynucleotidyl transfer-mediated dUTP nick end labeling (TUNEL analysis and 2,3,5-triphenyltetrazolium chloride staining. Tissue angiotensin II (Ang II and ACE activity were detected by enzyme-linked immunosorbent assays. The expression levels of ACE, Ang II type 1 receptor (AT1R, tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6, and interleukin-1β (IL-1β were measured by Western blot and real-time PCR. ACE inhibitory activity of Scu in vitro was detected by the photometric determination.Scu treatment dose-dependently decreased neurological deficit score, infarct area, cell apoptosis and morphological changes induced by pMCAO, which were associated with reductions of ACE and AT1R expression and the levels of Ang II, TNF-α, IL-6, and IL-1β in ischemic brains. Scu has a potent ACE inhibiting activity.Scu protects brain from acute ischemic injury probably through its inhibitory effect on the ACE/Ang II/AT1 axis, CBF preservation and proinflammation inhibition.

  7. Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

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    Gideon Hen

    Full Text Available The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV, into the chorioallantoic membrane (CAM of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP or recombinant alpha-melanocyte-stimulating hormone (α-MSH genes, driven by the cytomegalovirus (CMV promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP nick end labeling (TUNEL assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA, and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

  8. p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes)

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    Isoe, Yasuko; Okuyama, Teruhiro [Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Taniguchi, Yoshihito [Department of Preventive Medicine and Public Health, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Kubo, Takeo [Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Takeuchi, Hideaki, E-mail: takeuchi@biol.s.u-tokyo.ac.jp [Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Progenitor migration is accompanied by an increase in their numbers in the adult brain. Black-Right-Pointing-Pointer p53 Mutation suppressed an increase in the number of the migrated progenitors. Black-Right-Pointing-Pointer The decreased progenitor number is not due to enhanced cell death. Black-Right-Pointing-Pointer p53 Mutation did not affect proliferation of stem cells. -- Abstract: Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.

  9. Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa.

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    Domínguez-Rebolledo, A E; Fernández-Santos, M R; García-Alvarez, O; Maroto-Morales, A; Garde, J J; Martínez-Pastor, F

    2009-11-01

    The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 x g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1mM of the antioxidant Trolox, 100 microM of the oxidant Fe (with ascorbate), or both. The 3x4 treatments were incubated at 37 degrees C and assessed each hour up to 3h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4h was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30+/-3.52% to 67.94+/-5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.

  10. Extract of Azadirachta indica (Neem) Leaf Induces Apoptosis in 4T1 Breast Cancer BALB/c Mice.

    Science.gov (United States)

    Othman, Fauziah; Motalleb, Gholamreza; Lam Tsuey Peng, Sally; Rahmat, Asmah; Fakurazi, Sharida; Pei Pei, Chong

    2011-01-01

    Azadirachta indica (Neem) has been used traditionally for many centuries. Some impressive therapeutic qualities have been discovered. However, the therapeutic effect of neem leaf extract in 4T1 breast cancer has not been documented. The purpose of the present study is to investigate the therapeutic effect of ethanolic Neem leaf extract in an in vivo 4T1 breast cancer model in mice. A total of 84 female BALB/c mice were divided randomly into 7 groups (3 non-cancerous groups and 4 cancerous groups) consisting of 12 mice per group. The 3 non-cancerous groups were normal mice treated with 0.5% of Tween 20 in phosphate buffer saline (PBS) (NC), 250 mg/kg Neem (N250) or 500 mg/kg Neem (N500). The 4 cancerous groups were; cancer controls treated with 0.5% of Tween 20 in PBS (CC), and cancerous mice treated with 0.5 µg/mL tamoxifen citrate (CT), 250 mg/kg Neem leaf extract (CN 250) or 500 mg/kg Neem leaf extract (CN 500). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to evaluate apoptosis (cell death) in the breast cancer tissues. SPSS software, version 14 was used for statistical analysis. Statistical significance was defined as p≤0.05. Non parametric analysis of variance (ANOVA) was performed with the Kruskal Wallis test for the TUNEL assays. Parametric data among the groups was compared using ANOVA. TUNEL assays showed that the CN 250 and CN 500 groups had a higher incidence of apoptosis compared with the cancer controls. The findings showed that neem leaf extract induces apoptosis in 4T1 breast cancer BALB/c mice.

  11. Helicobacter pylori Infection in Association with Cell Proliferation,Apoptosis and Prostaglandin E2 Levels

    Institute of Scientific and Technical Information of China (English)

    PAN Kai-feng; ZHANG Yang; ZHANG Lian; MA Jun-ling; FENG Guo-shuang; ZHOU Tong; YOU Wei-cheng

    2007-01-01

    Objective: To evaluate the relationship between H. pylori infection with cell proliferation, apoptosis and PGE2 levels. Methods: A population-based study was conducted in Linqu, a high-risk area of gastric cancer in China. A total of 1523 subjects, aged 35-64, participating in a gastric cancer screening survey were investigated. H. pylori status were determined by 13C-urea breath test, expressions of Ki-67 were assessed by immunohistochemistry, apoptotic cells were detected by terminal deoxynucleotide transferase mediated dUTP nick end-labeling (TUNEL) method, and PGE2 levels were measured by enzyme immunoassay. Results: H. pylori infection was positively associated with cell proliferation activity. The mean and median percentage of Ki-67 labeling index (LI) in subjects with H. pylori positive were 14.1±10.3 and 12.0, significantly higher than those with H. pylori negative (-x±s: 8.4±7.0;median: 5.8;P<0.0001). Moreover, the prevalence rates of H. pylori infection showed a tendency to increase according to severity score of cell apoptosis (Ptrend <0.0001), from score 0 to 3, the percentage of H. pylori positivity increased from 67.5% to 96.7%. Furthermore, The mean and median of PGE2 concentration were 628.84±726.40 pg/mL and 411.33 pg/mL among subjects with H. pylori positive compared with 658.19±575.91pg/mL and 455.97 pg/mL among those with H. pylori negative (P=0.209). Conclusion: H. pylori infection was positively associated with increased cell proliferation and apoptosis activity, suggesting that H. pylori infection plays an important role in the gastric epithelial cell malignant transformation.

  12. Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification

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    Sayo Koike

    2016-09-01

    Full Text Available Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD. To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs stimulated calcium deposition in vascular smooth muscle cells (VSMCs through excessive oxidative stress and phenotypic transition into osteoblastic cells. Since AGEs can induce apoptosis, in this study we investigated its role on VSMC apoptosis, focusing mainly on the underlying mechanisms. A rat VSMC line (A7r5 was cultured, and treated with glycolaldehyde-derived AGE-bovine serum albumin (AGE3-BSA. Apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining. To quantify apoptosis, an enzyme-linked immunosorbent assay (ELISA for histone-complexed DNA fragments was employed. Real-time PCR was performed to determine the mRNA levels. Treatment of A7r5 cells with AGE3-BSA from 100 µg/mL concentration markedly increased apoptosis, which was suppressed by Nox inhibitors. AGE3-BSA significantly increased the mRNA expression of NAD(PH oxidase components including Nox4 and p22phox, and these findings were confirmed by protein levels using immunofluorescence. Dihydroethidisum assay showed that compared with cBSA, AGE3-BSA increased reactive oxygen species level in A7r5 cells. Furthermore, AGE3-induced apoptosis was significantly inhibited by siRNA-mediated knockdown of Nox4 or p22phox. Double knockdown of Nox4 and p22phox showed a similar inhibitory effect on apoptosis as single gene silencing. Thus, our results demonstrated that NAD(PH oxidase-derived oxidative stress are involved in AGEs-induced apoptosis of VSMCs. These findings might be important to understand the pathogenesis of vascular calcification in diabetes and CKD.

  13. Bioflavonoids Effects of Ginger on Glomerular Podocyte Apoptosis in Streptozotocin-Induced Diabetic Rat

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    Hajhosieni Laleh

    2014-04-01

    Full Text Available Objective: Ginger is a strong antioxidant and long-term treatment of streptozotocin (STZ-diabetic animals, and it has been shown to reduce oxidative stress. Prevalence oxidative stress among urban life and changes in antioxidant capacity are considered asplay an important role in the pathogenesis of chronic diabetes mellitus. Materials and Methods: Wistar male rat (n = 40 were divided into three groups, control group (n = 10 and Ginger Quercetin group that received 100 mg/kg (gavage, (n = 10, and diabetic group, which received 55 mg/kg intra peritoneal (IP STZ (n = 20, which was subdivided to two groups of 10; STZ group and treatment group. Treatment group received 55 mg/kg (IP STZ plus100 mg/kg ginger, daily for, 8 weeks, respectively; however, the control group just received an equal volume of distilled water daily (IP. Diabetes was induced by a single (IP injection of STZ (55 mg/kg. Animals were kept in standard condition. In 28 day after inducing diabetic 5 cc blood were collected for total antioxidant capacity, malondialdehyde and oxidized low density lipoprotein levels and kidney tissues of rat in whole groups were removed then prepared for apoptosis analysis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL method. Results: Apoptotic cells significantly decreased in group that has received 100 mg/kg ginger (P < 0.05 in comparison to experimental groups (P < 0.05. Conclusion: Since in our study 100 mg/kg ginger have significantly preventive effect on kidney cells damages by reducing number of apoptotic cells in kidney and hence it seems that using it can be effective for treatment in diabetic rat.

  14. Treadmill exercise ameliorates symptoms of Alzheimer disease through suppressing microglial activation-induced apoptosis in rats

    Science.gov (United States)

    Baek, Seung-Soo; Kim, Sang-Hoon

    2016-01-01

    Alzheimer disease (AD) is a most common form of dementia and eventually causes impairments of learning ability and memory function. In the present study, we investigated the effects of treadmill exercise on the symptoms of AD focusing on the microglial activation-induced apoptosis. AD was made by bilateral intracerebroventricular injection of streptozotocin. The rats in the exercise groups were made to run on a treadmill once a day for 30 min during 4 weeks. The distance and latency in the Morris water maze task and the latency in the step-down avoidance task were increased in the AD rats, in contrast, treadmill exercise shortened these parameters. The numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive and caspase-3-positive cells in the hippocampal dentate gyrus were decreased in the AD rats, in contrast, treadmill exercise suppressed these numbers. Expressions of glial fibrillary acidic protein (GFAP) and cluster of differentiation molecule 11B (CD11b) in the hippocampal dentate gyrus were increased in the AD rats, in contrast, treadmill exercise suppressed GFAP and CD11b expressions. Bax expression was increased and Bcl-2 expression was decreased in the hippocampus of AD rats, in contrast, treadmill exercise decreased Bax expression and increased Bcl-2 expression. The present results demonstrated that treadmill exercise ameliorated AD-induced impairments of spatial learning ability and short-term memory through suppressing apoptosis. The antiapoptotic effect of treadmill exercise might be ascribed to the inhibitory effect of treadmill exercise on microglial activation. PMID:28119873

  15. The neuroprotective role and mechanisms of TERT in neurons with oxygen-glucose deprivation.

    Science.gov (United States)

    Li, J; Qu, Y; Chen, D; Zhang, L; Zhao, F; Luo, L; Pan, L; Hua, J; Mu, D

    2013-11-12

    Telomerase reverse transcriptase (TERT) is reported to protect neurons from apoptosis induced by various stresses including hypoxia-ischemia (HI). However, the mechanisms by which TERT exerts its anti-apoptotic role in neurons with HI injury remain unclear. In this study, we examined the protective role and explored the possible mechanisms of TERT in neurons with HI injury in vitro. Primary cultured neurons were exposed to oxygen and glucose deprivation (OGD) for 3h followed by reperfusion to mimic HI injury in vivo. Plasmids containing TERT antisense, sense nucleotides, or mock were transduced into neurons at 48h before OGD. Expression and distribution of TERT were measured by immunofluorescence labeling and western blot. The expression of cleaved caspase 3 (CC3), Bcl-2 and Bax were detected by western blot. Neuronal apoptosis was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The mitochondrial reactive oxygen species (ROS) were measured by MitoSOX Red staining. Fluorescent probe JC-1 was used to measure the mitochondrial membrane potential (ΔΨm). We found that TERT expression increased at 8h and peaked at 24h in neurons after OGD. CC3 expression and neuronal apoptosis were induced and peaked at 24h after OGD. TERT inhibition significantly increased CC3 expression and neuronal apoptosis after OGD treatment. Additionally, TERT inhibition decreased the expression ratio of Bcl-2/Bax, and enhanced ROS production and ΔΨm dissipation after OGD. These data suggest that TERT plays a neuroprotective role via anti-apoptosis in neurons after OGD. The underlying mechanisms may be associated with regulating Bcl-2/Bax expression ratio, attenuating ROS generation, and increasing mitochondrial membrane potential.

  16. Astaxanthin alleviates oxidative stress insults-related derangements in human vascular endothelial cells exposed to glucose fluctuations.

    Science.gov (United States)

    Abdelzaher, Lobna A; Imaizumi, Takahiro; Suzuki, Tokiko; Tomita, Kengo; Takashina, Michinori; Hattori, Yuichi

    2016-04-01

    Glycemic fluctuations may play a critical role in the pathogenesis of diabetic complications, such as cardiovascular disease. We investigated whether the oxycarotenoid astaxanthin can reduce the detrimental effects of fluctuating glucose on vascular endothelial cells. Human umbilical venous endothelial cells were incubated for 3 days in media containing 5.5mM glucose, 22 mM glucose, or 5.5mM glucose alternating with 22 mM glucose in the absence or presence of astaxanthin or N-acetyl-L-cysteine (NAC). Constant high glucose increased reactive oxygen species (ROS) generation, but such an effect was more pronounced in fluctuating glucose. This was associated with up-regulated p22(phox) expression and down-regulated peroxisome proliferator activated receptor-γ coactivator (PGC-1α) expression. Astaxanthin inhibited ROS generation, p22(phox) up-regulation, and PGC-1α down-regulation by the stimuli of glucose fluctuation. Fluctuating glucose, but not constant high glucose, significantly decreased the endothelial nitric oxide synthase (eNOS) phosphorylation level at Ser-1177 without affecting total eNOS expression, which was prevented by astaxanthin as well as by the anti-oxidant NAC. Transferase-mediated dUTP nick end labeling (TUNEL) showed increased cell apoptosis in fluctuating glucose. Glucose fluctuation also resulted in up-regulating gene expression of pro-inflammatory mediators, interleukin-6 and intercellular adhesion molecule-1. These adverse changes were subdued by astaxanthin. The phosphorylation levels of c-Jun N-terminal kinase (JNK) and p38 were significantly increased by glucose fluctuations, and astaxanthin significantly inhibited the increase in JNK and p38 phosphorylation. Taken together, our results suggest that astaxanthin can protect vascular endothelial cells against glucose fluctuation by reducing ROS generation.

  17. Skeletal muscle DNA damage precedes spinal motor neuron DNA damage in a mouse model of Spinal Muscular Atrophy (SMA).

    Science.gov (United States)

    Fayzullina, Saniya; Martin, Lee J

    2014-01-01

    Spinal Muscular Atrophy (SMA) is a hereditary childhood disease that causes paralysis by progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. The mechanisms by which lack of SMN causes SMA pathology are not known, making it very difficult to develop effective therapies. We investigated whether DNA damage is a perinatal pathological event in SMA, and whether DNA damage and cell death first occur in skeletal muscle or spinal cord of SMA mice. We used a mouse model of severe SMA to ascertain the extent of cell death and DNA damage throughout the body of prenatal and newborn mice. SMA mice at birth (postnatal day 0) exhibited internucleosomal fragmentation in genomic DNA from hindlimb skeletal muscle, but not in genomic DNA from spinal cord. SMA mice at postnatal day 5, compared with littermate controls, exhibited increased apoptotic cell death profiles in skeletal muscle, by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling, and electron microscopy. SMA mice had no increased cell death, no loss of choline acetyl transferase (ChAT)-positive motor neurons, and no overt pathology in the ventral horn of the spinal cord. At embryonic days 13 and 15.5, SMA mice did not exhibit statistically significant increases in cell death profiles in spinal cord or skeletal muscle. Motor neuron numbers in the ventral horn, as identified by ChAT immunoreactivity, were comparable in SMA mice and control littermates at embryonic day 15.5 and postnatal day 5. These observations demonstrate that in SMA, disease in skeletal muscle emerges before pathology in spinal cord, including loss of motor neurons. Overall, this work identifies DNA damage and cell death in skeletal muscle as therapeutic targets for SMA.

  18. Beta-asarone induces LoVo colon cancer cell apoptosis by up-regulation of caspases through a mitochondrial pathway in vitro and in vivo.

    Science.gov (United States)

    Zou, Xi; Liu, Shen-Lin; Zhou, Jin-Yong; Wu, Jian; Ling, Bo-Fan; Wang, Rui-Ping

    2012-01-01

    Beta-asarone is one of the main bioactive constituents in traditional Chinese medicine Acorus calamu. Previous studies have shown that it has antifungal and anthelmintic activities. However, little is known about its anticancer effects. This study aimed to determine inhibitory effects on LoVo colon cancer cell proliferation and to clarify the underlying mechanisms in vitro and in vivo. Dose-response and time-course anti-proliferation effects were examined by MTT assay. Our results demonstrated that LoVo cell viability showed dose- and time-dependence on β-asarone. We further assessed anti-proliferation effects as β-asarone-induced apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide assay using a flow cytometer and observed characteristic nuclear fragmentation and chromatin condensation of apoptosis by microscopy. Moreover, we found the apoptosis to be induced through the mitochondrial/caspase pathway by decreasing mitochondrial membrane potential (MMP) and reducing the Bcl-2-to-Bax ratio, in addition to activating the caspase-9 and caspase-3 cascades. Additionally, the apoptosis could be inhibited by a pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). When nude mice bearing LoVo tumor xenografts were treated with β-asarone, tumor volumes were reduced and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays of excised tissue also demonstrated apoptotic changes. Taken together, these findings for the first time provide evidence that β-asarone can suppress the growth of colon cancer and the induced apoptosis is possibly mediated through mitochondria/caspase pathways.

  19. 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.

  20. Effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    杨林; 陶天遵; 王新婷; 杜宁; 陈伟珍; 陶树清; 王志成; 吴丽萍

    2003-01-01

    Objective To observe the effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro. Methods Iliac trabecular bone specimens were obtained from adult patients undergoing necessary surgery. After the bone pieces were digested with collagenase-trypsin, osteoblasts were released and incubated at 37℃ in a relative humidity of 95% and 5% CO2. Then, the cells were purified, and their passages were given DMEM-F12 and fetal bovine serum medium. Subsequently, 10-8 mol/L dexamethasone was added into the culture medium to incubate the osteoblasts for three days, and the cells from control groups were incubated without any drugs. All cells were observed continually with phase contrast microscope and transmission electron microscope. Finally, apoptosis was detected by the use of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and biochemical indices, alkaline phosphatase (ALP) and osteocalcin (OCN) were used to determine the effects of dexamethasone on proliferation, differentiation and apoptosis of adult osteoblasts in vitro. Results In the adult osteoblasts obtained by collagenase-trypsin digestion, it achieved high survial, stable biochemical indices and excellent purification. Under the condition of dexamethasone 10-8 mol/L and osteoblasts 10 000/ml, there was significant promotion of ALP and OCN secretion without cell apoptosis.Conclusions Dexamethasone has a significant effect on the proliferation and differentiation of adult osteoblasts in vitro without apoptosis, and dexamethasone at the suggested concentration can be used as positive control in drug studies for osteoporosis treatment.

  1. Is thioredoxin reductase involved in the defense against DNA fragmentation in varicocele?

    Institute of Scientific and Technical Information of China (English)

    Gül (o)zdemirler Erata; Canan Kü(c)ükgergin; Gülsan Aktan; Ates Kadioglu; Müjdat Uysal; Necla Ko(c)ak-Toker

    2013-01-01

    We aimed to investigate the role of thioredoxin reductase (TR) and inducible heat shock protein 70 (iHsp70) and their relationship with sperm quality in varicocele (VAR) patients.Semen samples were obtained from 16 subfertile men diagnosed as VAR and 10 fertile men who applied to the Andrology Laboratory of Istanbul Medical Faculty of Istanbul University.The sperm TR and iHsp 70 expression levels were determined using Western blot analysis.The TR activity of the sperm was assayed spectrophometrically.The sperm quality was evaluated both by conventional sperm analysis and by a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique that assayed DNA-fragmented spermatozoa in semen samples.The percentage of TUNEL-positive spermatozoa in the VAR group (16.3%±5.6%) was higher than that in the fertile group (5.5%± 1.9%).Significant inverse correlations were detected between the percentage of TUNEL-positive cells and both the concentration (r=-0.609; P=0.001) and motility (r=-0.550;P=0.004) of spermatozoa.Both the TR expression and activity were increased significantly in the VAR group (U=22.0; P=0.001 and U=33.5; P=0.012,respectively)as analyzed usingthe Mann-Whitney UWilcoxon rank sum Wtest.Furthermore,significant positive correlations were found between TR expression and activity (r=0.406; P=0.040) and between TR expression and the percentage of TUNEL-positive cells (r=0.665; P=0.001).Sperm iHsp70 expression did not differ between the VAR and fertile groups.In conclusion,increased sperm TR expression might be a defense mechanism against apoptosis in the spermatozoa of men with VAR.

  2. Combining Portable Raman Probes with Nanotubes for Theranostic Applications

    Directory of Open Access Journals (Sweden)

    Ashwinkumar A. Bhirde, Gang Liu, Albert Jin, Ramiro Iglesias-Bartolome, Alioscka A. Sousa, Richard D. Leapman, J. Silvio Gutkind, Seulki Lee, Xiaoyuan Chen

    2011-01-01

    Full Text Available Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs and multi-walled carbon nanotubes (MWCNTs were dispersed aqueously using a lipid-polymer (LP coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells

  3. Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase

    Institute of Scientific and Technical Information of China (English)

    Song Tusheng; Yang Ling; Huang Chen; Liu Liying; Ni Lei; Wang Aiying; Luo Yu

    2007-01-01

    Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid (IAA) at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase(HRP) at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate (DCFH-DA) uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. Results IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9%, which was 5 times that of control(P<0.01). Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

  4. Heat stress decreases testicular germ cell proliferation and increases apoptosis in short term: an immunohistochemical and ultrastructural study.

    Science.gov (United States)

    Kanter, Mehmet; Aktas, Cevat; Erboga, Mustafa

    2013-03-01

    Scrotal hyperthermia has been known as a cause of male infertility but the exact mechanism leading to impaired spermatogenesis is unknown. This work was aimed to investigate the role of scrotal hyperthermia on cell proliferation and apoptosis in testes. The rats were randomly allotted into one of the four experimental groups: A (control), B (1 day after scrotal hyperthermia), C (14 days after scrotal hyperthermia), and D (35 days after scrotal hyperthermia); each group comprised 7 animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for 6 consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia-exposed rats were killed under 50 mg/kg ketamine anaesthesia and tissue samples were obtained for biochemical and histopathological investigations. Hyperthermia treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione level, superoxide dismutase, and glutathione peroxidase activities. Moreover, exposure to hyperthermia resulted in lipid peroxidation increase in testes. Our data indicate a significant reduction in the expression of proliferating cell nuclear antigen and an enhancement in the activity of terminal deoxynucleotidyl transferase dUTP nick end labelling after scrotal hyperthermia. In scrotal hyperthermia, the mitochondrial degeneration, dilatation of smooth endoplasmic reticulum, and enlarged intercellular spaces were observed in both Sertoli and spermatid cells. Scrotal hyperthermia is one of the major factors that impair spermatogenesis in testis. This heat stress is shown to be closely associated with oxidative stress, followed by apoptosis of germ cells.

  5. In vitro antitumor properties of an isolate from leaves of Cassia alata L.

    Science.gov (United States)

    Olarte, Elizabeth Iglesias; Herrera, Annabelle Aliga; Villaseñor, Irene Manese; Jacinto, Sonia Donaldo

    2013-01-01

    Leaf extracts of Cassia alata L (akapulko), traditionally used for treatment of a variety of diseases, were evaluated for their potential antitumor properties in vitro. MTT assays were used to examine the cytotoxic effects of crude extracts on five human cancer cell lines, namely MCF-7, derived from a breast carcinoma, SK-BR-3, another breast carcinoma, T24 a bladder carcinoma, Col 2, a colorectal carcinoma, and A549, a non- small cell lung adenocarcinoma. Hexane extracts showed remarkable cytotoxicity against MCF-7, T24, and Col 2 in a dose-dependent manner. This observation was confirmed by morphological investigation using light microscopy. Further bioassay-directed fractionation of the cytotoxic extract led to the isolation of a TLC-pure isolate labeled as f6l. Isolate f6l was further evaluated using MTT assay and morphological and biochemical investigations, which likewise showed selectivity to MCF-7, T24, and Col 2 cells with IC50 values of 16, 17, and 17 μg/ml, respectively. Isolate f6l, however, showed no cytotoxicity towards the non-cancer Chinese hamster ovarian cell line (CHO-AA8). Cytochemical investigation using DAPI staining and biochemical investigation using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-a method used to detect DNA fragmentation-together with caspase assay, demonstrated apoptotic cell death. Spectral characterization of isolate f6l revealed that it contained polyunsaturated fatty acid esters. Considering the cytotoxicity profile and its mode of action, f6l might represent a new promising compound with potential for development as an anticancer drug with low or no toxicity to non-cancer cells used in this study.

  6. Maxadilan prevents apoptosis in iPS cells and shows no effects on the pluripotent state or karyotype.

    Directory of Open Access Journals (Sweden)

    Zhiyi Zhao

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a structurally endogenous peptide with many biological roles. Maxadilan, a 61-amino acid vasodilatory peptide, specifically activates the PACAP type I receptor (PAC1. Although PAC1 has been identified in embryonic stem cells, little is known about its presence or effects in human induced pluripotent stem (iPS cells. In the present study, we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR and western blot analysis. To study the physiological effects mediated by PAC1, we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC. After exposure to UVC, the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells, as demonstrated by WST-8 analysis, annexin V/propidium iodide (PI analysis and the terminal transferase dUTP nick end labeling (TUNEL assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly, immunofluorescence, western blot analysis, real-time quantitative polymerase chain reaction (RT-qPCR analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover, karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary, these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype.

  7. Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

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    Rahman HS

    2014-01-01

    Full Text Available Heshu Sulaiman Rahman,1–3 Abdullah Rasedee,1,2 Ahmad Bustamam Abdul,2,4 Nazariah Allaudin Zeenathul,1,2 Hemn Hassan Othman,1,3 Swee Keong Yeap,2 Chee Wun How,2 Wan Abd Ghani Wan Nor Hafiza4,51Faculty of Veterinary Medicine, 2Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia; 3Faculty of Veterinary Medicine, University of Sulaimanyah, Sulaimanyah City, Kurdistan Region, Northern Iraq; 4Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor, Malaysia; 5College of Medical Laboratory Technology, Institute for Medical Research, Kuala Lumpur, MalaysiaAbstract: This investigation evaluated the antileukemia properties of a zerumbone (ZER-loaded nanostructured lipid carrier (NLC prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl-2,5 diphenyltetrazolium bromide assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose polymerase (PARP. These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans.Keywords: zerumbone-loaded nanostructured lipid carrier, cell cycle arrest, apoptosis, mitochondrial pathway

  8. Cytotoxic Effects of GM1 Ganglioside and Amyloid β-Peptide on Mouse Embryonic Neural Stem Cells

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    Makoto Yanagisawa

    2010-01-01

    Full Text Available AD (Alzheimer's disease is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between GM1 ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells, but the precise effects of GM1 and Aβ on NSCs have not yet been clarified. We evaluated the effect of GM1 and Aβ-(1–40 on mouse NECs (neuroepithelial cells, which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either GM1 or Aβ-(1–40. On the contrary, a decreased number of NECs were cultured in the presence of a combination of GM1 and Aβ-(1–40. The exogenously added GM1 and Aβ-(1–40 were confirmed to incorporate into NECs. The Ras–MAPK (mitogen-activated protein kinase pathway, important for cell proliferation, was intact in NECs simultaneously treated with GM1 and Aβ-(1–40, but caspase 3 was activated. NECs treated with GM1 and Aβ-(1–40 were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, an indicator of cell death. It was found that GM1 and Aβ-(1–40 interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1–40 and GM1 co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.

  9. Anti-apoptotic effect of dexamethasone in an ototoxicity model.

    Science.gov (United States)

    Lee, Jin Ho; Oh, Se Heang; Kim, Tae Ho; Go, Yoon Young; Song, Jae-Jun

    2017-01-01

    Dexamethasone (DEX) is used for the treatment of various inner ear diseases. However, the molecular mechanism of DEX on gentamicin induced hair cell damage is not known. Therefore, this study investigated the protective effect of DEX on gentamicin (GM)-induced ototoxicity and the effect of GM on the expression of apoptosis related genes. The protective effects of DEX were measured by phalloidin staining of explant cultures of organ of Corti from postnatal day 2-3 mice with GM-induced hair cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to detect apoptosis and immunofluorescence was done to analyze the effect of DEX on the expression of apoptosis related genes. Cochlear explant cultures of postnatal day-4-old mice were exposed to 0, 1, 5, 10, 30, 50, and 100 μg/ml DEX and GM during culture. DEX protected from GM-induced hair cell loss in the inner ear of postnatal day 4 mice. To understand the molecular mechanisms by which DEX pre-treatment decreased hair cell loss, the testes of cochlear explant cultures of postnatal day 4 mice were examined for changes in expression of cochlear apoptosis mediators. The pro-apoptotic protein Bax was significantly down-regulated and numbers of apoptotic hair cells were decreased. DEX has a protective effect on GM-induced hair cell loss in neonatal cochlea cultures and the protective mechanism may involve inhibition of the mitochondrial apoptosis pathway. The combination with scaffold technique can improve delivery of DEX into the inner ear to protect GM-induced ototoxicity.

  10. Protective Effect of Tempol against Cisplatin-Induced Ototoxicity.

    Science.gov (United States)

    Youn, Cha Kyung; Kim, Jun; Jo, Eu-Ri; Oh, Jeonghyun; Do, Nam Yong; Cho, Sung Il

    2016-11-18

    One of the major adverse effects of cisplatin chemotherapy is hearing loss. Cisplatin-induced ototoxicity hampers treatment because it often necessitates dose reduction, which decreases cisplatin efficacy. This study was performed to investigate the effect of Tempol on cisplatin-induced ototoxicity in an auditory cell line, House Ear Institute-Organ of Corti 1 (HEI-OC1). Cultured HEI-OC1 cells were exposed to 30 μM cisplatin for 24 h with or without a 2 h pre-treatment with Tempol. Cell viability was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and apoptotic cells were identified using terminal deoxynucleotidyl transferase dUTP nick end labeling of nuclei (TUNEL) assay and flow cytometry. The effects of Tempol on cisplatin-induced cleaved poly(ADP-ribose) polymerase, cleaved caspase, and mitochondrial inducible nitric oxide synthase expression were evaluated using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured to assess the effects of Tempol on cisplatin-induced ROS accumulation. Mitochondria were evaluated by confocal microscopy, and the mitochondrial membrane potential was measured to investigate whether Tempol protected against cisplatin-induced mitochondrial dysfunction. Cisplatin treatment decreased cell viability, and increased apoptotic features and markers, ROS accumulation, and mitochondrial dysfunction. Tempol pre-treatment before cisplatin exposure significantly inhibited all these cisplatin-induced effects. These results demonstrate that Tempol inhibits cisplatin-induced cytotoxicity in HEI-OC1, and could play a preventive role against cisplatin-induced ototoxicity.

  11. Using basic fibroblast growth factor nanoliposome combined with ultrasound-introduced technology to early intervene the diabetic cardiomyopathy.

    Science.gov (United States)

    Zhao, Ying-Zheng; Zhang, Ming; Tian, Xin-Qiao; Zheng, Lei; Lu, Cui-Tao

    2016-01-01

    Basic fibroblast growth factor (bFGF)-loaded liposome (bFGF-lip) combined with ultrasound-targeted microbubble destruction (UTMD) technique was investigated to prevent diabetic cardiomyopathy (DCM). Cardiac function and myocardial ultrastructure were assessed. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, immunohistochemistry staining, and Western blot assay were used to investigate the signal pathway underlying the expression of bFGF in DCM treatment. From Mason staining and TUNEL staining, bFGF-lip + UTMD group showed significant differences from the diabetes group and other groups treated with bFGF or bFGF-lip. The diabetes group showed similar results (myocardial capillary density, collagen volume fraction, and cardiac myocyte apoptosis index) to other bFGF treatment groups. Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis, and metabolic disturbances could be suppressed by the different extents of twice-weekly bFGF treatments for 12 consecutive weeks (free bFGF or bFGF-lip +/- UTMD), with the strongest improvements observed in the bFGF-lip + UTMD group. The group combining bFGF-lip with UTMD demonstrated the highest level of bFGF expression among all the groups. The bFGF activated the PI3K/AKT signal pathway, causing the reduction of myocardial cell apoptosis and increase of microvascular density. This strategy using bFGF-lip and UTMD is a potential strategy in early intervention of DCM in diabetes.

  12. Fabrication of novel vesicles of triptolide for antirheumatoid activity with reduced toxicity in vitro and in vivo.

    Science.gov (United States)

    Zhang, Li; Wang, Tengteng; Li, Qiang; Huang, Jing; Xu, Hao; Li, Jinlong; Wang, Yongjun; Liang, Qianqian

    2016-01-01

    Triptolide (TP) displays a strong immunosuppression function in immune-mediated diseases, especially in the treatment of rheumatoid arthritis. However, in addition to its medical and health-related functions, TP also exhibits diverse pharmacological side effects, for instance, liver and kidney toxicity and myelosuppression. In order to reduce the side effects, a nano drug carrier system (γ-PGA-l-PAE-TP [PPT]), in which TP was loaded by a poly-γ-glutamic acid-grafted l-phenylalanine ethylester copolymer, was developed. PPT was characterized by photon scattering correlation spectroscopy and transmission electron microscopy, which demonstrated that the average diameter of the drug carrier system is 98±15 nm, the polydispersity index is 0.18, the zeta potential is -35 mV, and the TP encapsulation efficiency is 48.6% with a controlled release manner. The methylthiazolyldiphenyl-tetrazolium bromide assay and flow cytometry revealed that PPT could decrease toxicity and apoptosis induced by free TP on RAW264.7 cells, respectively. The detection of reactive oxygen species showed that PPT could decrease the cellular reactive oxygen species induced by TP. Compared with the free TP-treated group, PPT improved the survival rate of the mice (P0.05) and spleen index (P>0.05). The blood biochemical indexes revealed that PPT did not cause much damage to the kidney (blood urea nitrogen and creatinine), liver (serum alanine aminotransferase and aspartate aminotransferase), or blood cells (P>0.05). Meanwhile, hematoxylin and eosin staining and terminal-deoxynucleotidyl transferase dUTP nick-end labeling staining indicated that PPT reduced the damage of free TP on the liver, kidney, and spleen. Our results demonstrated that PPT reduced free TP toxicity in vitro and in vivo and that it is a promising fundamental drug delivery system for rheumatoid arthritis treatment.

  13. Aluminium-Induced Oxidative Stress, Apoptosis and Alterations in Testicular Tissue and Sperm Quality in Wistar Rats: Ameliorative Effects of Curcumin

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    Ebrahim Cheraghi

    2017-09-01

    Full Text Available Background Reproductive toxicity is a major challenge associated with aluminum (Al exposure. No studies have evaluated the possible effects of curcumin (CUR on Al-induced reproductive dysfunction. Therefore, this study investigated the effects of CUR treatment on Al-induced reproductive damage. Materials and Methods In this experimental study, 40 male Wistar rats were allocated to the five groups (n=8 based on the treatment they received: no treatment (control, solvent [dimethyl sulfoxide (DMSO or distilled water], CUR 10 mg/kg body weight (BW, Al chloride 10 mg/kg BW, and CUR+Al chloride (10 mg/kg BW/each alone. Treatments were performed by intraperitoneal (IP injections for 28 days. The left testis was assessed for histopathological analysis as well as the incidence of germ cell apoptosis. One-way analysis of variance (ANOVA followed by the Tukey’s test was used. P<0.05 was considered significant. A value of P<0.05 was considered significant. Results Significant reductions in body and testis weight; plasma testosterone and luteinizing hormone levels; sperm count, motility, morphology, and viability; germinal epithelium thickness; seminiferous tubules diameter; as well as, superoxide dismutase activity were observed in rats treated with Al. Moreover, Al exposure caused significant increments in the lumen diameter of tubules, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL-positive cells and malondialdehyde (MDA levels compared to the control group. However, in rats receiving CUR+Al, CUR significantly reversed the adverse effects of Al on testis and sperm quality. No significant differences in follicle-stimulating hormone (FSH levels and nuclear diameter of spermatogonia were detected among all groups. Conclusion It can be concluded that Al causes reproductive dysfunction by creating oxidative damage. CUR, on the other hand, reduces the toxic effects of Al and improves the antioxidant status and sperm quality in male rats.

  14. Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer.

    LENUS (Irish Health Repository)

    Bennett, M W

    2012-02-03

    BACKGROUND: Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. AIM: To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. SPECIMENS: Thirty paraffin wax embedded human gastric adenocarcinomas. METHODS: FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. CONCLUSIONS: Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.

  15. The Fas counterattack in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinoma.

    LENUS (Irish Health Repository)

    Bennett, M W

    2012-02-03

    Various cancer cell lines express Fas ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression has been demonstrated in several human malignancies in vivo. We sought to determine whether human esophageal carcinomas express FasL, and whether FasL expression is associated with increased apoptosis of tumor-infiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Using in situ hybridization and immunohistochemistry, respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n = 6; adenocarcinoma, n = 2). The Extent of FasL expression was variable, with both FasL-positive and FasL-negative neoplastic regions occurring within tumors. TIL were detected by immunohistochemical staining for the leukocyte common Ag, CD45. FasL expression was associated with a mean fourfold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6- to 12-fold, n = 6,p < 0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean twofold increase in detectable cell death among TIL in FasL-positive areas compared with FasL-negative areas (range 1.6- to 2.4-fold, n = 6, p < 0.05). In conclusion, we demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest Fas-mediated apoptotic depletion of TIL in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer.

  16. Therapeutic effects of high molecular weight hyaluronan injections for tendinopathy in a rat model.

    Science.gov (United States)

    Yoshida, Mamoru; Funasaki, Hiroki; Kubota, Makoto; Marumo, Keishi

    2015-01-01

    Tendinopathy is the most common tendon disorder. The etiology is still uncertain, and the disorder poses many therapeutic problems. In a few clinical studies, analgesic effects of high molecular weight hyaluronan (HMW HA) injections were observed, but the underlying mechanisms were not elucidated. In the present study, we analyzed the therapeutic effects of hyaluronan injections for tendinopathy in an animal model. We made the tendinopathy rat model using a rodent treadmill machine. Rats with tendinopathy were injected with HMW HA (HA group), normal saline (NS group), or nothing (control group) into the space between the patellar tendon and the fat pad bilaterally, or were injected with HMW HA into the right knees and with saline to the left knees (HA/NS group), 5 times every 4 days. To assess the pain-relieving effect of HA, the spontaneous locomotor activities at night (12 h) and weight bearing of hind paws were measured every day. Histological sections of the patellar tendon stained with hematoxylin-eosin or prepared by TdT-mediated dUTP nick end labeling were microscopically analyzed. The number of spontaneous locomotor activities in the HA group was significantly larger than those in NS or control groups, and in the HA group they recovered up to a healthy level. The percent weight distribution of the right hind paws was significantly increased along with the number of injections. On histologic examinations, the numbers of microtears, laminations, or apoptotic cells in the patellar tendons in the HA group were significantly lower than those in the NS or the control groups. The injections of HMW HA were effective for pain relief and for partial restoration of the patellar tendon in our tendinopathy rat model, and thus may become an effective therapeutic modality for the disease.

  17. Prokaryotic expression and action mechanism of antimicrobial LsGRP1(C) recombinant protein containing a fusion partner of small ubiquitin-like modifier.

    Science.gov (United States)

    Lin, Chia-Hua; Pan, Ying-Chieh; Liu, Fang-Wei; Chen, Chao-Ying

    2017-09-30

    Antimicrobial peptides (AMPs) are peptides exhibiting broad-spectrum antimicrobial activities and considered as potential therapeutic agents. LsGRP1(C), a novel AMP derived from defense-related LsGRP1 protein of Lilium, was proven to inhibit kinds of bacteria and fungi via alteration of microbial membrane permeability and induction of fungal programmed cell death-like phenomena by in vitro assays using synthetic LsGRP1(C). In this study, the prokaryotic production of LsGRP1(C) recombinant protein containing an N-terminal fusion partner of the yeast small ubiquitin-like modifier (SUMO) was achieved by using optimized Escherichia coli host and purification buffer system, which lead to a high yield of soluble SUMO-LsGRP1(C) fusion protein. In vitro assay revealed that E. coli-expressed SUMO-LsGRP1(C) exhibited even better antifungal activity as compared to synthetic LsGRP1(C). Meanwhile, the ability of SUMO-LsGRP1(C) in conducting fungal membrane permeabilization and programmed cell death was verified by SYTOX Green staining and 4',6-diamidino-2-phenylindole staining/terminal deoxynucleotidyl transferase dUTP nick-end labeling assays, respectively, indicating that E. coli-expressed SUMO-LsGRP1(C) shares identical modes of action with synthetic LsGRP1(C). Herein, this E. coli expression system enables the effective and convenient production of antimicrobial LsGRP1(C) in a form of SUMO-fused recombinant protein.

  18. Experimental research on recombinant human endostatin-induced cardiomyocyte apoptosis in rats

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    Jing QIN

    2014-03-01

    Full Text Available Objective To explore the recombinant human endostatin (rh-ES-induced cardiotoxicity in rats and its mechanism. Methods Twenty four female Wistar rats were randomly divided into four groups (6 each. Rats in low, moderate and high dose group received rh-ES with a dosage of 3, 6 and 12mg/(kg·d, respectively, by intraperitoneal injection, and rats in control group received the same amount of normal saline alone. Half of rats in each group were sacrificed by spinal dislocation after 4 weeks and 8 weeks of the treatment. Pathomorphologic and ultrastructural changes in rat's myocardial tissue were evaluated by light microscopy and transmission electron microscopy. Cardiomyocyte apoptosis was detected with TdT-mediated dUTP nick end labeling (TUNEL assay. Microvessel density (MVD in myocardial tissue was measured by immunohistochemically marking endothelial cell with CD34. Results No pathomorphologic and ultrastrucural changes were found under light microscope and transmission electron microscope in the low dose and moderate dose groups, but cardiomyocyte damage were found in the high dose group. TUNEL assay revealed more apoptotic cells in high and moderate (only 8 weeks dose groups than in control group (P=0.033, P=0.000, and the apoptosis index was highest in the high dose group at 8 weeks. In addition, compared with the control group, MVD significantly increased in high dose groups at 4 weeks and 8 weeks (P<0.05. Conclusions rh-ES induces the cardiotoxicity in rats, and cardiomyocyte apoptosis is involved in the pathological course of cardiac toxicity. DOI: 10.11855/j.issn.0577-7402.2014.01.02

  19. Acute doxorubicin insult in the mouse ovary is cell- and follicle-type dependent.

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    Elon C Roti Roti

    Full Text Available Primary ovarian insufficiency (POI is one of the many unintended consequences of chemotherapy faced by the growing number of female cancer survivors. While ovarian repercussions of chemotherapy have long been recognized, the acute insult phase and primary sites of damage are not well-studied, hampering efforts to design effective intervention therapies to protect the ovary. Utilizing doxorubicin (DXR as a model chemotherapy agent, we defined the acute timeline for drug accumulation, induced DNA damage, and subsequent cellular and follicular demise in the mouse ovary. DXR accumulated first in the core ovarian stroma cells, then redistributed outwards into the cortex and follicles in a time-dependent manner, without further increase in total ovarian drug levels after four hours post-injection. Consistent with early drug accumulation and intimate interactions with the blood supply, stroma cell-enriched populations exhibited an earlier DNA damage response (measurable at 2 hours than granulosa cells (measurable at 4 hours, as quantified by the comet assay. Granulosa cell-enriched populations were more sensitive however, responding with greater levels of DNA damage. The oocyte DNA damage response was delayed, and not measurable above background until 10-12 hours post-DXR injection. By 8 hours post-DXR injection and prior to the oocyte DNA damage response, the number of primary, secondary, and antral follicles exhibiting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive granulosa cells plateaued, indicating late-stage apoptosis and suggesting damage to the oocytes is subsequent to somatic cell failure. Primordial follicles accumulate significant DXR by 4 hours post-injection, but do not exhibit TUNEL-positive granulosa cells until 48 hours post-injection, indicating delayed demise. Taken together, the data suggest effective intervention therapies designed to protect the ovary from chemotherapy accumulation and induced insult

  20. Assessment of Retrograde Coronary Venous Infusion of Mesenchymal Stem Cells Combined with Basic Fibroblast Growth Factor in Canine Myocardial Infarction Using Strain Values Derived from Speckle-Tracking Echocardiography.

    Science.gov (United States)

    Sun, Qi-Wei; Zhen, Lei; Wang, Qin; Sun, Yan; Yang, Jiao; Li, Yi-Jia; Li, Rong-Juan; Ma, Ning; Li, Zhi-An; Wang, Lu-Ya; Nie, Shao-Ping; Yang, Ya

    2016-01-01

    Speckle-tracking echocardiography was used to assess retrograde coronary venous infusion of mesenchymal stem cells (MSCs) combined with basic fibroblast growth factor (bFGF) in a canine model of acute myocardial infarction (AMI). AMI was induced by ligation of the left anterior descending coronary artery. Coronary venous retroperfusion was performed at 1 wk after AMI. Twenty-eight animals were randomized into four groups: saline, bFGF+saline, saline+MSCs and bFGF+MSCs. Echocardiography was performed before AMI, at 7 d post-AMI and 40 d after retroperfusion. Apoptotic cardiomyocytes in the border zone of the ischemic region were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Vascular endothelial growth factor and factor VIII concentrations were measured by western blotting. The left ventricular end-systolic volume increased significantly, whereas the left ventricular ejection fraction and global and segmental strain values decreased significantly after AMI. After retroperfusion, the strain values of the infarct zone, but not conventional echocardiographic parameters, were significantly different between control and bFGF+MSC groups. Cardiomyocyte apoptosis decreased, whereas vascular endothelial growth factor and factor VIII concentrations were higher in the bFGF+MSC, bFGF and MSC groups. Cardiomyocyte apoptosis was well correlated with the strain values. Although retrograde coronary venous infusion of bFGF and MSCs promoted neo-vascularization of the infarcted myocardium and inhibited apoptosis, there was only a slight strain improvement without a substantial increase in global cardiac functions.

  1. Continuous lactation effects on mammary remodeling during late gestation and lactation in dairy goats.

    Science.gov (United States)

    Safayi, S; Theil, P K; Hou, L; Engbaek, M; Nørgaard, J V; Sejrsen, K; Nielsen, M O

    2010-01-01

    The present study aimed to 1) elucidate whether continuous milking during late gestation in dairy goats negatively affects mammary remodeling and hence milk production in the subsequent lactation, and 2) identify the regulatory factors responsible for changes in cell turnover and angiogenesis in the continuously lactating mammary gland. Nine multiparous dairy goats were used. One udder half was dried off approximately 9 wk prepartum (normal lactation; NL), and the other udder half of the same goat was milked continuously (continuous lactation; CL) until parturition or until the half-udder milk yields had dropped to below 50 g/d. Mammary biopsies were obtained from each udder half just before the NL gland was dried off (before dry period), within the first 2 wk after drying-off (early dry period, samples available only for NL glands), in the mid dry period, within the last 2 wk before parturition (late dry period), and at d 1 (the day of parturition), 3, 10, 60, and 180 of lactation. Mammary morphology was characterized in biopsies by quantitative histology, and cell turnover was determined by immunohistochemistry (terminal deoxynucleotidyl transferase dUTP nick end labeling and Ki-67). Transcription of genes encoding factors involved in mammary epithelial cell (MEC) turnover and vascular function was quantified by quantitative reverse transcription PCR. Results demonstrated that omitting the dry period was possible in goats but was not as easy as claimed before. Renewal of MEC was suppressed in CL glands, which resulted in a smaller MEC population in the subsequent lactation. At the time of parturition (and throughout lactation), the mammary glands subjected to CL had smaller alveoli, more fully differentiated MEC, and a substantially larger capillary fraction compared with NL glands. The continuously lactating gland thus resembled a normally lactating gland in an advanced stage of lactation. None of the studied genomic factors could account for these treatment

  2. Alpha lipoic acid protects lens from H2O2-induced cataract by inhibiting apoptosis of lens epithelial cells and inducing activation of anti-oxidative enzymes

    Institute of Scientific and Technical Information of China (English)

    Yun Li; Ya-Zhen Liu; Jing-Ming Shi; Song-Bai Jia

    2013-01-01

    Objective: To determine whether alpha lipoic acid (LA) can effectively protect lenses from hydrogen peroxide (H2O2)-induced cataract. Methods: Lens from adult Sprague-Dawley rats were cultured in 24-well plates and treated without or with 0.2 mM of H2O2, 0.2 mM of H2O2 plus 0.5 mM, 1.0 mM, or 2.0 mM of LA for 24 h. Cataract was assessed using cross line grey scale measurement. Superoxide dismutase (SOD), glutathione (GSH-Px), lactate dehydrogenase (LDH), and malondialdehyde (MDA) activity or level in lens homogenates was measured. Apoptosis of lens epithelial cells in each group were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay. Results: A total of 0.2 mM of H2O2 induced obvious cataract formation and apoptosis in lens’ epithelial cells, but 0.5-2.0 mM of LA could block the effect of 0.2 mM H2O2 in inducing cataract and apoptosis. Furthermore, 0.2 mM of H2O2 significantly decreased SOD, GSH-Px, and LDH activity and significant increased MDA level in the lens, but 0.5-2.0 mM of LA blocked the effect of 0.2 mM H2O2. One mM of LA was found to be the most effective. Conclusions: LA can protect lens from H2O2-induced cataract. LA exerts protective effects through inhibition of lens’ epithelial cell apoptosis and activation of anti-oxidative enzymes.

  3. Programmed cell death in kiwifruit stigmatic arms and its relationship to the effective pollination period and the progamic phase.

    Science.gov (United States)

    Ferradás, Yolanda; López, Marián; Rey, Manuel; González, Ma Victoria

    2014-07-01

    Kiwifruit is a crop with a highly successful reproductive performance, which is impaired by the short effective pollination period of female flowers. This study investigates whether the degenerative processes observed in both pollinated and non-pollinated flowers after anthesis may be considered to be programmed cell death (PCD). Features of PCD in kiwifruit, Actinidia chinensis var. deliciosa, were studied in both non-pollinated and pollinated stigmatic arms using transmission electron microscopy, DAPI (4',6-diamidino-2-phenylindole) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assays, DNA gel electrophoresis and caspase-like activity assays. In the secretory tissues of the stigmatic arms, cell organelles disintegrated sequentially while progressive vacuolization was detected. At the same time, chromatin condensation, nuclear deformation, and DNA fragmentation and degradation were observed. These features were detected in both non-pollinated and pollinated stigmatic arms; they were evident in the stigmas of pollinated flowers by the second day after anthesis but only by 4 d after anthesis in non-pollinated flowers. In addition, in pollinated stigmatic arms, these features were first initiated in the stigma and gradually progressed through the style, consistent with pollen tube growth. This timing of events was also observed in both non-pollinated and pollinated stigmatic arms for caspase-3-like activity. The data provide evidence to support the hypothesis that PCD processes occurring in the secretory tissue of non-pollinated kiwifruit stigmatic arms could be the origin for the observed short effective pollination period. The results obtained in the secretory tissue of pollinated kiwifruit stigmatic arms upon pollination support the idea that PCD might be accelerated by pollination, pointing to the involvement of PCD during the progamic phase. © The Author 2014. Published by Oxford University Press on behalf of the

  4. Inhibition of human breast cancer xenograft growth by cruciferous vegetable constituent benzyl isothiocyanate.

    Science.gov (United States)

    Warin, Renaud; Xiao, Dong; Arlotti, Julie A; Bommareddy, Ajay; Singh, Shivendra V

    2010-05-01

    Benzyl isothiocyanate (BITC), a constituent of cruciferous vegetables such as garden cress, inhibits growth of human breast cancer cell lines in culture. The present study was undertaken to determine in vivo efficacy of BITC against MDA-MB-231 human breast cancer xenografts. The BITC administration retarded growth of MDA-MB-231 cells subcutaneously implanted in female nude mice without causing weight loss or any other side effects. The BITC-mediated suppression of MDA-MB-231 xenograft growth correlated with reduced cell proliferation as revealed by immunohistochemical analysis for Ki-67 expression. Analysis of the vasculature in the tumors from BITC-treated mice indicated smaller vessel area compared with control tumors based on immunohistochemistry for angiogenesis marker CD31. The BITC-mediated inhibition of angiogenesis in vivo correlated with downregulation of vascular endothelial growth factor (VEGF) receptor 2 protein levels in the tumor. Consistent with these results, BITC treatment suppressed VEGF secretion and VEGF receptor 2 protein levels in cultured MDA-MB-231 cells. Moreover, the BITC-treated MDA-MB-231 cells exhibited reduced capacity for migration compared with vehicle-treated control cells. In contrast to cellular data, BITC administration failed to elicit apoptotic response as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. In conclusion, the present study demonstrates in vivo anti-cancer efficacy of BITC against MDA-MB-231 xenografts in association with reduced cell proliferation and suppression of neovascularization. These preclinical observations merit clinical investigation to determine efficacy of BITC against human breast cancers.

  5. Infliximab protects against pulmonary emphysema in smoking rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiang-yan; HAN Jing; ZHANG Cheng; SUN Qian-yun; LI Dan; LUO Rong-rong; WAN Zi-fen; YE Xian-wei; LIU Wei-jia; RAO Shan-shan

    2011-01-01

    Background It is widely accepted that tumor necrosis factor-α (TNF-a) plays an important role in the pathogenesis of emphysema. This study aimed at investigating the protective effects of anti-TNF-α antibody, infliximab, in the development of emphysema induced by passive smoking in rats.Methods Thirty-nine rats were randomly divided into a normal control group (group 1), an emphysema group (group 2),and an infliximab-intervention group (group 3). Rat models of emphysema were established by exposure to cigarette smoking daily for 74 days. After 1 month, the infliximab intervention group was treated with infliximab via subcutaneous injection. The levels of TNF-α, iL-8 and vascular endothelial growth factor (VEGF) in bronchoalveolar lavage fluid (BALF)were measured with enzyme linked immunosorbent assay (ELISA). The number and classification of cells in the BALF were measured. Lung tissue sections stained by hematoxylin and eosin (HE) were observed, and mean linear intercept (MLI) and mean alveolar numbers (MAN) were measured. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods were used to examine the percentage of positive cells and distribution of apoptotic cells.Results The levels of TNF-α and IL-8 in BALF were higher in group 2 than in group 1 and group 3. The MLI was greater in group 2 than that in group 1 and group 3 while MAN was decreased. The concentration of VEGF in BALF of group 2 was significantly decreased as compared with group 1. The total cells and neutrophils number was significantly increased in group 2 as compared with group 1 and group 3, so was the percentage of neutrophils. The number of TUNEL positive cells in the alveolar septa was significantly increased in group 2 as compared with group 1 and group 3.Conclusion Infliximab protects against cigarette smoking-induced emphysema by reducing airway inflammation,attenuating alveolar septa cell apoptosis and improving pathological changes.

  6. Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis

    Science.gov (United States)

    Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

    2017-01-01

    Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1

  7. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation.

    Science.gov (United States)

    Pedrotty, Dawn M; Koh, Jennifer; Davis, Bryce H; Taylor, Doris A; Wolf, Patrick; Niklason, Laura E

    2005-04-01

    Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled us to separate the three-dimensional (3-D) electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblasts were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and not differences in apoptosis, as determined by proliferating cell nuclear antigen and TdT-mediated dUTP nick-end labeling. Cardiomyocyte-conditioned medium also significantly increased myoblast proliferation. Expression of transcription factors governing differentiation along skeletal or cardiac lineages was evaluated by immunoblotting. Although these assays are qualitative, no changes in differentiation state along skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of skeletal myoblasts. Hence, cardiac milieu appears to stimulate proliferation but does not affect differentiation of skeletal myoblasts.

  8. Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-β, PDGF-BB, c-Kit and SCF genes

    Science.gov (United States)

    Kadivar, Ali; Kamalidehghan, Behnam; Akbari Javar, Hamid; Karimi, Benyamin; Sedghi, Reihaneh; Noordin, Mohamed Ibrahim

    2017-01-01

    Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-β and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-β, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2–10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-β, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer. PMID:28260860

  9. Hydrogen Gas Inhalation Attenuates Seawater Instillation-Induced Acute Lung Injury via the Nrf2 Pathway in Rabbits.

    Science.gov (United States)

    Diao, Mengyuan; Zhang, Sheng; Wu, Lifeng; Huan, Le; Huang, Fenglou; Cui, Yunliang; Lin, Zhaofen

    2016-12-01

    Seawater instillation-induced acute lung injury involves oxidative stress and apoptosis. Although hydrogen gas inhalation is reportedly protective in multiple types of lung injury, the effect of hydrogen gas inhalation on seawater instillation-induced acute lung injury remains unknown. This study investigated the effect of hydrogen gas on seawater instillation-induced acute lung injury and explored the mechanisms involved. Rabbits were randomly assigned to control, hydrogen (2 % hydrogen gas inhalation), seawater (3 mL/kg seawater instillation), and seawater + hydrogen (3 mL/kg seawater instillation + 2 % hydrogen gas inhalation) groups. Arterial partial oxygen pressure and lung wet/dry weight ratio were detected. Protein content in bronchoalveolar lavage fluid (BALF) and serum as well as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 levels were determined. Hematoxylin-eosin staining was used to monitor changes in lung specimens, and malondialdehyde (MDA) content and myeloperoxidase (MPO) activity were assayed. In addition, NF-E2-related factor (Nrf) 2 and heme oxygenase (HO)-1 mRNA and protein expression were measured, and apoptosis was assessed by measuring caspase-3 expression and using terminal deoxy-nucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. Hydrogen gas inhalation markedly improved lung endothelial permeability and decreased both MDA content and MPO activity in lung tissue; these changes were associated with decreases in TNF-α, IL-1β, and IL-6 in BALF. Hydrogen gas also alleviated histopathological changes and cell apoptosis. Moreover, Nrf2 and HO-1 expressions were significantly activated and caspase-3 expression was inhibited. These results demonstrate that hydrogen gas inhalation attenuates seawater instillation-induced acute lung injury in rabbits and that the protective effects observed may be related to the activation of the Nrf2 pathway.

  10. In Vitro Ultramorphological Assessment of Apoptosis on CEMss Induced by Linoleic Acid-Rich Fraction from Typhonium flagelliforme Tuber

    Directory of Open Access Journals (Sweden)

    Syam Mohan

    2011-01-01

    Full Text Available The plant Typhonium flagelliforme, commonly known as “rodent tuber” in Malaysia, is often used as a health supplement and traditional remedy for alternative cancer therapies, including leukemia. This study aimed to evaluate in vitro anti-leukemic activity of dichloromethane extract/fraction number 7 (DCM/F7 from T. flagelliforme tuber on human T4 lymphoblastoid (CEMss cell line. The DCM extract of tuber has been fractionated by column chromatography. The obtained fractions were evaluated for its cytotoxicity toward CEMss cells as well as human primary blood lymphocytes (PBLs. Assessment of apoptosis produced by the most active fraction was evaluated by various microscopic techniques and further confirmation of apoptosis was done by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. Phytochemical screening was done by gas chromatography-mass spectrometry (GC-MS. The results shows that 7 out of 12 fractions showed significant cytotoxicity against the selected cell line CEMss, in which fractions DCM/F7, DCM/F11 and DCM/F12 showed exceptional activity with 3, 5 and 6.2 μg ml−1, respectively. Further studies in the non-cancerous PBL exhibited significant selectivity of DCM/F7 compared to other fractions. Cytological observations showed chromatin condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double-staining of acridine orange (AO/propidium iodide (PI, SEM and TEM. In addition, DCM/F7 has increased the cellular DNA breaks on treated cells. GC-MS revealed that DCM/F7 contains linoleic acid, hexadecanoic acid and 9-hexadecanoic acid. The present results indicate that T. flagelliforme possess a valuable anti-leukemic effect and was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

  11. In Vitro Ultramorphological Assessment of Apoptosis on CEMss Induced by Linoleic Acid-Rich Fraction from Typhonium flagelliforme Tuber.

    Science.gov (United States)

    Mohan, Syam; Bustamam, Ahmad; Ibrahim, Siddig; Al-Zubairi, Adel S; Aspollah, Mohammed; Abdullah, Rasedee; Elhassan, Manal Mohamed

    2011-01-01

    The plant Typhonium flagelliforme, commonly known as "rodent tuber" in Malaysia, is often used as a health supplement and traditional remedy for alternative cancer therapies, including leukemia. This study aimed to evaluate in vitro anti-leukemic activity of dichloromethane extract/fraction number 7 (DCM/F7) from T. flagelliforme tuber on human T4 lymphoblastoid (CEMss) cell line. The DCM extract of tuber has been fractionated by column chromatography. The obtained fractions were evaluated for its cytotoxicity toward CEMss cells as well as human primary blood lymphocytes (PBLs). Assessment of apoptosis produced by the most active fraction was evaluated by various microscopic techniques and further confirmation of apoptosis was done by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Phytochemical screening was done by gas chromatography-mass spectrometry (GC-MS). The results shows that 7 out of 12 fractions showed significant cytotoxicity against the selected cell line CEMss, in which fractions DCM/F7, DCM/F11 and DCM/F12 showed exceptional activity with 3, 5 and 6.2 μg ml(-1), respectively. Further studies in the non-cancerous PBL exhibited significant selectivity of DCM/F7 compared to other fractions. Cytological observations showed chromatin condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double-staining of acridine orange (AO)/propidium iodide (PI), SEM and TEM. In addition, DCM/F7 has increased the cellular DNA breaks on treated cells. GC-MS revealed that DCM/F7 contains linoleic acid, hexadecanoic acid and 9-hexadecanoic acid. The present results indicate that T. flagelliforme possess a valuable anti-leukemic effect and was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

  12. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Institute of Scientific and Technical Information of China (English)

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  13. 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.

  14. Anatase titanium dioxide nanoparticles in mice: evidence for induced structural and functional sperm defects after short-, but not long-, term exposure.

    Science.gov (United States)

    Smith, Michelle A; Michael, Rowan; Aravindan, Rolands G; Dash, Soma; Shah, Syed I; Galileo, Deni S; Martin-DeLeon, Patricia A

    2015-01-01

    Titanium dioxide (TiO 2 ) nanoparticles (TNPs) are widely used commercially and exist in a variety of products. To determine if anatase TNPs (ATNPs) in doses smaller than previously used reach the scrotum after entry in the body at a distant location and induce sperm defects, 100% ATNP (2.5 or 5 mg kg-1 body weight) was administered intraperitoneally to adult males for three consecutive days, followed by sacrifice 1, 2, 3, or 5 weeks later (long-) or 24, 48 or 120 h (short-term exposure). Transmission electron microscopy revealed the presence of ANTP in scrotal adipose tissues collected 120 h postinjection when cytokine evaluation showed an inflammatory response in epididymal tissues and fluid. At 120 h and up to 3 weeks postinjection, testicular histology revealed enlarged interstitial spaces. Significantly increased numbers of terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling-positive (apoptotic) germ (P = 0.002) and interstitial space cells (P = 0.04) were detected in treated males. Caudal epididymal sperm from the short-term, but not a long-term, arm showed significantly (P < 0.001) increased frequencies of flagellar abnormalities, excess residual cytoplasm (ERC), and unreacted acrosomes in treated versus controls (dose-response relationship). A novel correlation between ERC and unreacted acrosomes was uncovered. At 120 h, there were significant decreases in hyperactivated motility (P < 0.001) and mitochondrial membrane potential (P < 0.05), and increased reactive oxygen species levels (P < 0.00001) in treated versus control sperm. These results indicate that at 4-8 days postinjection, ANTP induce structural and functional sperm defects associated with infertility, and DNA damage via oxidative stress. Sperm defects were transient as they were not detected 10 days to 5 weeks postinjection.

  15. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1.

    Science.gov (United States)

    Yang, Jiong; Yao, Shukun

    2015-10-27

    Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC). The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1) matrine not only activated caspase and PARP (poly ADP-ribose polymerase) cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2) matrine treatment promoted the JNK-Bcl-2/ Bcl-xL-Bax/Bak pathway; (3) Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4) Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5) finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1.

  16. Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

    Directory of Open Access Journals (Sweden)

    Fu Na

    2010-10-01

    Full Text Available Abstract Objective Heme oxygenase-1 (HO-1, the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant properties. However, the role of HO-1 on hepatocyte apoptosis remains unclear. We aim to elucidate the effects of HO-1 on oxidative stress related hepatocellular apoptosis in nutritional steatohepatitis in mice. Methods C57BL/6J mice were fed with methionine-choline deficient (MCD diet for four weeks to induce hepatic steatohepatitis. HO-1 chemical inducer (hemin, HO-1 chemical inhibitor zinc protoporphyrin IX (ZnPP-IX and/or adenovirus carrying HO-1 gene (Ad-HO-1 were administered to mice, respectively. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay, the mRNA and protein expression of apoptosis related genes were assayed by quantitative real-time PCR and Western blot. Results Hepatocyte signs of oxidative related apoptotic injury were presented in mice fed with MCD diet for 4 weeks. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver histology, which was associated with decreased hepatic lipid peroxidation content, reduced number of apoptotic cells by TUNEL staining, down-regulated expression of pro-apoptosis related genes including Fas/FasL, Bax, caspase-3 and caspase-9, reduced expression of cytochrome p4502E1 (CYP2E1, inhibited cytochrome c (Cyt-c release, and up-regulated expression of anti-apoptosis gene Bcl-2. Whereas, inhibition of HO-1 by ZnPP-IX caused oxidative stress related hepatic injury, which concomitant with increased number of TUNEL positive cells and up-regulated expression of pro-apoptosis related genes. Conclusions The present study provided evidences for the protective role of HO-1 in preventing nutritional steatohepatitis through suppressing hepatocyte apoptosis in mice.

  17. A hexane fraction of American ginseng suppresses mouse colitis and associated colon cancer: anti-inflammatory and proapoptotic mechanisms.

    Science.gov (United States)

    Poudyal, Deepak; Le, Phuong Mai; Davis, Tia; Hofseth, Anne B; Chumanevich, Alena; Chumanevich, Alexander A; Wargovich, Michael J; Nagarkatti, Mitzi; Nagarkatti, Prakash S; Windust, Anthony; Hofseth, Lorne J

    2012-04-01

    Ulcerative colitis is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American ginseng extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of American ginseng that suppress colitis and prevent colon cancer. Among five different fractions of American ginseng (butanol, hexane, ethylacetate, dichloromethane, and water), a hexane fraction has particularly potent antioxidant and proapoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex vivo model (CD4(+)/CD25(-) primary effector T cells). A key in vivo finding was that compared with the whole American ginseng extract, the hexane fraction of American ginseng was more potent in treating colitis in a dextran sodium sulfate (DSS) mouse model, as well as suppressing azoxymethane/DSS-induced colon cancer. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling of inflammatory cells within the colonic mesenteric lymph nodes was elevated in mice consuming DSS + the hexane fraction of American ginseng. Results are consistent with our in vitro data and with the hypothesis that the hexane fraction of American ginseng has anti-inflammatory properties and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of American ginseng that suppress colitis and prevent colon cancer associated with colitis.

  18. AT1R blocker losartan attenuates intestinal epithelial cell apoptosis in a mouse model of Crohn's disease.

    Science.gov (United States)

    Liu, Tian-Jing; Shi, Yong-Yan; Wang, En-Bo; Zhu, Tong; Zhao, Qun

    2016-02-01

    Angiotensin II, which is the main effector of the renin‑angiotensin system, has an important role in intestinal inflammation via the angiotensin II type 1 receptor (AT1R). The present study aimed to investigate the protective effects of the AT1R blocker losartan on 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis. Losartan was administered to male adult C57BL/6 J mice 2 weeks prior to the induction of colitis, and images of the whole colon were captured to record changes, scored according to a microscopic scoring system, and reverse transcription-quantitative polymerase chain reaction were performed in order to investigate colonic inflammation. In addition, intestinal epithelial barrier permeability was evaluated, and intestinal epithelial cell (IEC) apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and apoptosis-related protein expression levels were detected by western blotting. Losartan was able to attenuate TNBS-induced body weight loss and colonic damage. Furthermore, T helper 1-mediated proinflammatory cytokines were suppressed by losartan, and gut permeability was largely preserved. TUNEL staining revealed reduced IEC apoptosis in the losartan-treated mice. Losartan also increased the B-cell lymphoma 2 (Bcl2)/Bcl-2-associated X protein (Bax) ratio and suppressed caspase-3 induction. These results suggested that the AT1R blocker losartan may attenuate TNBS-induced colitis by inhibiting the apoptosis of IECs. The effects of losartan were partially mediated through increasing the Bcl-2/Bax ratio and subsequently suppressing the induction of the proapoptotic mediator caspase-3.

  19. Imidacloprid impairs the post-embryonic development of the midgut in the yellow fever mosquito Stegomyia aegypti (=Aedes aegypti).

    Science.gov (United States)

    Fernandes, K M; Gonzaga, W G; Pascini, T V; Miranda, F R; Tomé, H V V; Serrão, J E; Martins, G F

    2015-09-01

    The mosquito Stegomyia aegypti (=Aedes aegypti) (Diptera: Culicidae) is a vector for the dengue and yellow fever viruses. As blood digestion occurs in the midgut, this organ constitutes the route of entry of many pathogens. The effects of the insecticide imidacloprid on the survival of St. aegypti were investigated and the sub-lethal effects of the insecticide on midgut development were determined. Third instar larvae were exposed to different concentrations of imidacloprid (0.15, 1.5, 3.0, 6.0 and 15.0 p.p.m.) and survival was monitored every 24 h for 10 days. Midguts from imidacloprid-treated insects at different stages of development were dissected and processed for analyses by transmission electron microscopy, immunofluorescence microscopy and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays. Imidacloprid concentrations of 3.0 and 15.0 p.p.m. were found to affect midgut development similarly. Digestive cells of the fourth instar larvae (L4) midgut exposed to imidacloprid had more multilamellar bodies, abundantly found in the cell apex, and more electron-lucent vacuoles in the basal region compared with those from untreated insects. Moreover, imidacloprid interfered with the differentiation of regenerative cells, dramatically reducing the number of digestive and endocrine cells and leading to malformation of the midgut epithelium in adults. The data demonstrate that imidacloprid can reduce the survival of mosquitoes and thus indicate its potentially high efficacy in the control of St. aegypti populations.

  20. Effect of L-glutamine on liver Bcl-2 mRNA expression after total hepatic inflow occlusion in rats%谷氨酰胺对大鼠肝门阻断后肝脏Bcl-2 mRNA表达的影响及其保护作用

    Institute of Scientific and Technical Information of China (English)

    刘国平; 朱闻溪; 杨广顺; 周文平; 程广明

    2008-01-01

    目的:探讨谷氨酰胺(L-glutamine,Gln)对肝门阻断后肝脏细胞凋亡及Bcl-2 mRNA表达的影晌.方法:雄性Wistar大鼠,随机分为假手术组(A组)、对照组(B组)和实验组(C组)3组.采用Pringle's法进行肝门阻断,持续35 min,肝门阻断前C组大鼠腹腔注射Gln.分别于肝门阻断前及再灌注后2、4和24 h,每组各选取10只大鼠,测定血清ALT、AST、乳酸脱氢酶(lactic dehydrogenase,LDH)含量;检测肝组织谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)的含量;采用原位末端脱氧核苷酸转移酶法(terminal deoxynucleotidyl transferase-mediated DUTP nick end labeling method,TUNEL)检测肝脏细胞凋亡,并计算凋亡指数(apoptosic index,Al);采用RT-PCR方法检测肝Bcl-2 mRNA的表达.结果:与B组相比,再灌注后C组肝组织中MDA含量下降(P<0.05),而GSH水平增高(P<0.05);血清ALT、AST、LDH含量及Al均明显降低(P<0.05);Bcl-2mRNA表达则显著增强(P<0.05).结论:Gln能够减轻过氧化损伤,上调肝脏Bcl-2 mRNA的表达,抑制肝细胞凋亡,从而在肝门阻断中发挥保护作用.

  1. THE EXPERIMENTAL STUDY ON THE CELL APOPTOSIS AND EXPRESSION OF BCL-2 PROTEIN IN INTRACEREBRAL HEMORRHAGE IN MODEL OF RATS

    Institute of Scientific and Technical Information of China (English)

    Bao Gang; Guo Ning; Zhang Zhonglin; Chen Wei; Bao Dehu

    2006-01-01

    Otjective To study whether there is the apoptosis of neural cells and the expressionof Bcl-2 protein in intracerebral hemorrhage (ICH) in model of rats, for the further understanding the mechanism of the delayed damage of the neural cells around the hematoma after ICH. Methods Fifty SD rats were randomly divided into 5 groups, ten in each. With the Group A as the control, the rest 40 were used to set up intracerebral hemorrhage model. The brains were taken out at 12th, 24th, 48th and 72th hours, respectively. Apoptosis cells were detected with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), and the expression of Bcl-2 protein was detected with immunochemical stainging methed (SP). Results In the control group, no apoptosis cells and Bcl-2protein were detected. In rest groups, the apoptosis cells and Bcl-2 protein were expressed in different degree.Apoptosis rates verified and corresponded with the time after ICH, with the peak at 48th -72th hour after hemorrhage.The peak rate of apoptosis cells was (24. 50± 2.69)% and Bcl-2 protein expression was (20. 76 ± 1.97)% . There was significant difference between the experimental groups and control (P<0.05), and no linear relationship between the apoptosis rate and the expression of Bcl-2 protein. Conclusion Apoptosis may be an important factor in the secondary trauma of ICH. There is a time leg after hemorrhage. All this is instructive to clinical treatment in time. Bcl-2 protein keeps increasing in a certain time after hemorrhage, but not synchronize with the cell apoptosis. This indicates that bcl-2 has the effect to reduce the apoptosis of neural cells.

  2. Proliferation, migration, and differentiation of endog