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Sample records for deoxynucleotidyltransferase-mediated dutp nick

  1. Analysis of Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting Using Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry.

    Science.gov (United States)

    Yang, Jing-Feng; Gao, Rong-Chun; Wu, Hai-Tao; Li, Peng-Fei; Hu, Xian-Shu; Zhou, Da-Yong; Zhu, Bei-Wei; Su, Yi-Cheng

    2015-11-04

    The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm(2) and then held at room temperature for melting development. The samples were histologically processed into formalin-fixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity.

  2. Nick Laws | NREL

    Science.gov (United States)

    -3238 Nick's primary work at NREL revolves around REopt - a techno-economic optimization model for , Nick is continuously adapting the REopt model to accommodate the latest technologies, market factors spiral and the impact of utility rate structures on the adoption of residential solar photovoltaics and

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

    DEFF Research Database (Denmark)

    Nielsen, Ida; Andersen, Anni Hangaard; Bjergbæk, Lotte

    2012-01-01

    The Flp-nick system is a simple in vivo system developed for studying the cellular responses to a protein-bound nick at a single genomic site in the budding yeast Saccharomyces cerevisiae. The Flp-nick system takes advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp ...

  4. 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...

  5. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

    NARCIS (Netherlands)

    Vriend, Lianne E. M.; Prakash, Rohit; Chen, Chun-Chin; Vanoli, Fabio; Cavallo, Francesca; Zhang, Yu; Jasin, Maria; Krawczyk, Przemek M.

    2016-01-01

    DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided

  6. 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.

  7. Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses

    International Nuclear Information System (INIS)

    Hu, Xiaolan; Yu, Yingnian; Li, Qian; Wu, Danxiao; Tan, Zhengning; Wang, Cheng; Wang, Jvping; Wu, Meiping

    2011-01-01

    Highlights: → MNNG-induced appearance of DUT-N in the extracellular fluid has cellular specificity. → MNNG alters the subcellular distribution of DUT-N in human cells in different ways. → DUT-N may be a potential biomarker to assess the risk of alkylating agents exposure. -- Abstract: Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25 μM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.

  8. 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)

  9. 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.

  10. Bypass of a Nick by the Replisome of Bacteriophage T7*

    Science.gov (United States)

    Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C.

    2011-01-01

    DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase·polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick. PMID:21701044

  11. Bypass of a nick by the replisome of bacteriophage T7.

    Science.gov (United States)

    Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C

    2011-08-12

    DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.

  12. 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.

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

    International Nuclear Information System (INIS)

    Shoji, Wataru; Suenaga, Yusuke; Kaneko, Yoshiki; Islam, S.M. Rafiqul; Alagu, Jennifer; Yokoi, Sana; Nio, Masaki; Nakagawara, Akira

    2015-01-01

    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

  14. 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.

  15. 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

  16. Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

    Science.gov (United States)

    Odell, M; Shuman, S

    1999-05-14

    The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.

  17. 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

  18. An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification.

    Science.gov (United States)

    Lin, Zhenyu; Yang, Weiqiang; Zhang, Guiyun; Liu, Qida; Qiu, Bin; Cai, Zongwei; Chen, Guonan

    2011-08-28

    A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.

  19. Securing wilderness landscapes in South Africa : Nick Steele, private wildlife conservancies and saving rhinos

    NARCIS (Netherlands)

    Wels,; H.,

    2015-01-01

    Private wildlife conservation is booming business in South Africa! Nick Steele stood at the cradle of this development in the politically turbulent 1970s and 1980s, by stimulating farmers in Natal (now KwaZulu-Natal) to pool resources in order to restore wilderness landscapes, but at the same time

  20. 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

  1. Extensive immune-mediated hippocampal damage in mice surviving infection with neuroadapted Sindbis virus

    International Nuclear Information System (INIS)

    Kimura, Takashi; Griffin, Diane E.

    2003-01-01

    Viral infections of the central nervous system and immune responses to these infections cause a variety of neurological diseases. Infection of weanling mice with Sindbis virus causes acute nonfatal encephalomyelitis followed by clearance of infectious virus, but persistence of viral RNA. Infection with a neuroadapted strain of Sindbis virus (NSV) causes fatal encephalomyelitis, but passive transfer of immune serum after infection protects from fatal disease and infectious virus is cleared. To determine whether persistent NSV RNA is associated with neurological damage, we examined the brains of recovered mice and found progressive loss of the hippocampal gyrus, adjacent white matter, and deep cerebral cortex associated with mononuclear cell infiltration. Mice deficient in CD4 + T cells showed less tissue loss, while mice lacking CD8 + T cells showed lesions comparable to those in immunocompetent mice. Mice deficient in both CD4 + and CD8 + T cells developed severe tissue loss similar to immunocompetent mice and this was associated with extensive infiltration of macrophages. The number of CD4 + cells and macrophage/microglial cells, but not CD8 + cells, infiltrating the hippocampal gyrus was correlated with the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive pyramidal neurons. These results suggest that CD4 + T cells can promote progressive neuronal death and tissue injury, despite clearance of infectious virus

  2. 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.

  3. 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.

  4. Nick translation detection in situ of cellular DNA strand break induced by radiation

    International Nuclear Information System (INIS)

    Maehara, Y.; Anai, H.; Kusumoto, T.; Sakaguchi, Y.; Sugimachi, K.

    1989-01-01

    DNA strand break in HeLa cells induced by radiation was detected using the in situ nick translation method. The cells were exposed to radiation of 3, 6, 12, 18, and 24 Gy in Lab-Tek tissue culture chamber/slides and were fixed with ethanol/acetic acid on the slide glass. The break sites in DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [ 3 H]-labeled dTTP. Autoradiographic observation was made of the level of break sites in the DNA. The DNA strand break appeared even with a 3 Gy exposure, increased 8.6 times at 24 Gy compared with the control cells, and this level correlated reciprocally to change in cell viability. This nick translation method provides a rapid in situ assay for determining radiation-induced DNA damage of cultured cells, in a semi-quantitative manner

  5. 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

  6. Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

    Science.gov (United States)

    Conacci-Sorrell, Maralice; Ngouenet, Celine; Eisenman, Robert N

    2010-08-06

    The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation. Copyright 2010 Elsevier Inc. All rights reserved.

  7. The singer and the song: Nick Cave and the archetypal function of the cover version

    OpenAIRE

    Wiseman-Trowse, Nathan J B

    2013-01-01

    Throughout his career, from The Boys Next Door, through The Birthday Party, and with The Bad Seeds, Australian singer / songwriter Nick Cave has balanced his own set of creative voices alongside those of others through his choice of cover versions. Cave’s 1986 album with The Bad Seeds, ‘Kicking Against the Pricks’, is a collection of cover versions that spans American folk idioms (‘Black Betty’, ‘Hey Joe’, ‘The Singer’), Tin-Pan-Alley balladeering (‘Something’s Gotten Hold of my Heart’, ‘By t...

  8. Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain.

    Science.gov (United States)

    Musi, Valeria; Spolaore, Barbara; Picotti, Paola; Zambonin, Marcello; De Filippis, Vincenzo; Fontana, Angelo

    2004-05-25

    Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the

  9. Preparation and Characterization of Nicke-iron Alloy Film as Freestanding Electrode for Oxygen Evolution Reaction

    Directory of Open Access Journals (Sweden)

    Yao Mengqi

    2018-01-01

    Full Text Available This work reports the porous nicke-iron alloy film supported on stainless steel mesh as freestanding electrode for enhanced oxygen evolution reaction (OER catalyst prepared from an one step electrodeposition method. Results indicated that the porous nickle-iron alloy film exhibits a low overpotential of 270 mV at 10 mA cm-2 and excellent electroconductibility. The superior OER properties can be attributed to its novel synthetic process, conductive substrate and porous structure. This work will provide a new strategy to fabricate alloy film for OER electrocatalyst.

  10. Dehalogenation of chloroalkanes by nickel(i) porphyrin derivatives, a computational study.

    Science.gov (United States)

    Szatkowski, L; Hall, M B

    2016-11-14

    The nickel(i) octaethylisobacteriochlorin anion ([OEiBCh-Ni (I) ] - ) is commonly used as a synthetic model of cofactor F 430 from Methyl-Coenzyme M Reductase. In this regard, experimental studies show that [OEiBCh-Ni (I) ] - can catalyze dehalogenation of aliphatic halides in DMF solution by a highly efficient S N 2 reaction. To better understand this process, we constructed theoretical models of the dehalogenation of chloromethane by a simple nickel(i) isobacteriochlorin anion and compared its reactivity with that of similar Ni (I) complexes with other porphyrin-derived ligands: porphyrin, chlorin, bactreriochlorin, hexahydroporphyrin and octahydroporphyrin. Our calculations predict that all of the porphyrin derivative's model reactions proceed through low-spin complexes. Relative to the energy of the separate reactants the theoretical activation energies (free-energy barriers with solvation corrections) for the dehalogenation of chloromethane are similar for all of the porphyrin derivatives and range for the different functionals from 10-15 kcal mol -1 for B3LYP to 5-10 kcal mol -1 for M06-L and to 13-18 kcal mol -1 for ωB97X-D. The relative free energies of the products of the dehalogenation step, L-Ni-Me adducts, have a range from -5 to -40 kcal mol -1 for all functionals; generally becoming more negative with increasing saturation of the porphyrin ligand. Moreover, no significant differences in the theoretical chlorine kinetic isotope effect were discernable with change of porphyrin ligand.

  11. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. 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.

  13. Processive nicking activity of T4 endonuclease V on UV-irradiated chromatin

    International Nuclear Information System (INIS)

    Gruskin, E.A.; Lloyd, R.S.

    1986-01-01

    T4 endonuclease V initiates the excision repair of pyrimidine dimers in UV-irradiated T4 infected E. coli cells. The pyrimidine dimer specific nicking activity of T4 endonuclease V functions by a processive scanning on UV-irradiated DNA. Previously it has been demonstrated that introduction of endonuclease V into repair-deficient human cells causes a restoration of UV survival in these cells. This demonstrates that endonuclease V is competent to incise mammalian DNA at the site of pyrimidine dimers. In order to assess the ability of endonuclease V to act processively on DNA associated as chromatin, minichromosomes were prepared for use as a substrate. Form I DNA was reconstituted with H3, H4 +/- H1 histones by sequential dialysis steps from 2.0 M NaCl to 50 mM NaCl. Time course reactions were performed with minichromosomes containing 10 and 25 dimers per molecule. In each case the rate of disappearance of form I DNA which was associated as chromatin was decreased relative to that of naked form I DNA. Concurrent with that observation, the rate and extent of appearance of form III DNA was increased with the DNA in minichromosomes relative to naked DNA. This is diagnostic of an enhancement of processivity. The inclusion of H1 in the minichromosomes resulted in a slight additional increase in processivity relative to minichromosomes consisting only of H3 and H4

  14. An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

    Science.gov (United States)

    Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

    2018-05-15

    An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Last stop on the road to repair: structure of E. coli DNA ligase bound to nicked DNA-adenylate.

    Science.gov (United States)

    Nandakumar, Jayakrishnan; Nair, Pravin A; Shuman, Stewart

    2007-04-27

    NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria and essential for growth. Their distinctive substrate specificity and domain organization vis-a-vis human ATP-dependent ligases make them outstanding targets for anti-infective drug discovery. We report here the 2.3 A crystal structure of Escherichia coli LigA bound to an adenylylated nick, which captures LigA in a state poised for strand closure and reveals the basis for nick recognition. LigA envelopes the DNA within a protein clamp. Large protein domain movements and remodeling of the active site orchestrate progression through the three chemical steps of the ligation reaction. The structure inspires a strategy for inhibitor design.

  16. Rhombic-Shaped Nanostructures and Mechanical Properties of 2D DNA Origami Constructed with Different Crossover/Nick Designs.

    Science.gov (United States)

    Ma, Zhipeng; Huang, Yunfei; Park, Seongsu; Kawai, Kentaro; Kim, Do-Nyun; Hirai, Yoshikazu; Tsuchiya, Toshiyuki; Yamada, Hirofumi; Tabata, Osamu

    2018-01-01

    DNA origami methods enable the fabrication of various nanostructures and nanodevices, but their effective use depends on an understanding of their structural and mechanical properties and the effects of basic structural features. Frequency-modulation atomic force microscopy is introduced to directly characterize, in aqueous solution, the crossover regions of sets of 2D DNA origami based on different crossover/nick designs. Rhombic-shaped nanostructures formed under the influence of flexible crossovers placed between DNA helices are observed in DNA origami incorporating crossovers every 3, 4, or 6 DNA turns. The bending rigidity of crossovers is determined to be only one-third of that of the DNA helix, based on interhelical electrostatic forces reported elsewhere, and the measured pitches of the 3-turn crossover design rhombic-shaped nanostructures undergoing negligible bending. To evaluate the robustness of their structural integrity, they are intentionally and simultaneously stressed using force-controlled atomic force microscopy. DNA crossovers are verified to have a stabilizing effect on the structural robustness, while the nicks have an opposite effect. The structural and mechanical properties of DNA origami and the effects of crossovers and nicks revealed in this paper can provide information essential for the design of versatile DNA origami structures that exhibit specified and desirable properties. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. 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.

  18. Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa

    Science.gov (United States)

    Rebello, George; Ramesar, Rajkumar; Vorster, Alvera; Roberts, Lisa; Ehrenreich, Liezle; Oppon, Ekow; Gama, Dumisani; Bardien, Soraya; Greenberg, Jacquie; Bonapace, Giuseppe; Waheed, Abdul; Shah, Gul N.; Sly, William S.

    2004-01-01

    Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa. PMID:15090652

  19. Expression of proteinase inhibitor II proteins during floral development in Solanum americanum.

    Science.gov (United States)

    Sin, Suk-Fong; Chye, Mee-Len

    2004-10-01

    The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

  20. Alpha-Adrenoceptor Antagonists Improve Memory by Activating -methyl-D-Aspartate-Induced Ion Currents in the Rat Hippocampus

    Directory of Open Access Journals (Sweden)

    Chang Hee Kim

    2015-12-01

    Full Text Available Purpose: Alpha1 (α1-adrenoceptor antagonists are widely used to treat lower urinary tract symptoms. These drugs not only act on peripheral tissues, but also cross the blood-brain barrier and affect the central nervous system. Therefore, α1-adrenoceptor antagonists may enhance brain functions. In the present study, we investigated the effects of tamsulosin, an α1-adrenoceptor antagonist, on short-term memory, as well as spatial learning and memory, in rats. Methods: The step-down avoidance test was used to evaluate short-term memory, and an eight-arm radial maze test was used to evaluate spatial learning and memory. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining was performed in order to evaluate the effect of tamsulosin on apoptosis in the hippocampal dentate gyrus. Patch clamp recordings were used to evaluate the effect of tamsulosin on ionotropic glutamate receptors, such as N-methyl-D-aspartate (NMDA, amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA, and kainate receptors, in hippocampal CA1 neurons. Results: Tamsulosin treatment improved short-term memory, as well as spatial learning and memory, without altering apoptosis. The amplitudes of NMDA-induced ion currents were dose-dependently increased by tamsulosin. However, the amplitudes of AMPA- and kainate-induced ion currents were not affected by tamsulosin. Conclusions: Tamsulosin enhanced memory function by activating NMDA receptor-mediated ion currents in the hippocampus without initiating apoptosis. The present study suggests the possibility of using tamsulosin to enhance memory under normal conditions, in addition to its use in treating overactive bladder.

  1. Double-tracer autoradiography with Cu-ATSM/FDG and immunohistochemical interpretation in four different mouse implanted tumor models

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Takeshi [Department of Otorhinolaryngology, University of Fukui, Matsuoka, Eiheiji-cho, Yoshida-gun, Fukui 910-1193 (Japan)]. E-mail: wplants@mac.com; Furukawa, Takako [Biomedical Imaging Research Center, University of Fukui, Matsuoka, Eiheiji-cho, Yoshida-gun, Fukui 910-1193 (Japan); Fujieda, Shigeharu [Department of Otorhinolaryngology, University of Fukui, Matsuoka, Eiheiji-cho, Yoshida-gun, Fukui 910-1193 (Japan); Kasamatsu, Shingo [Biomedical Imaging Research Center, University of Fukui, Matsuoka, Eiheiji-cho, Yoshida-gun, Fukui 910-1193 (Japan); Yonekura, Yoshiharu [Biomedical Imaging Research Center, University of Fukui, Matsuoka, Eiheiji-cho, Yoshida-gun, Fukui 910-1193 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, Matsuoka, Eiheiji-cho, Yoshida-gun, Fukui 910-1193 (Japan)]. E-mail: yfuji@fmsrsa.fukui-med.ac.jp

    2006-08-15

    Background: We studied the regional characteristics within tumor masses using PET tracers and immunohistochemical methods. Methods: The intratumoral distribution of {sup 64}Cu-diacetyl-bis(N4-methylthiosemicarbazone) ([{sup 64}Cu]Cu-ATSM) and [{sup 18}F] 2-fluoro-2-deoxyglucose ({sup 18}F]FDG) in mice with tumors of four different origins (LLC1, Meth-A, B16 and colon26) was compared with the immunohistochemical staining of proliferating cells (Ki67), blood vessels (CD34 or von Willebrand factor), and apoptotic cells (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling method). Results: With all cell lines, [{sup 64}Cu]Cu-ATSM and [{sup 18}F]FDG were distributed with different gradation in the tumor mass. The immunohistochemical study demonstrated that the high [{sup 64}Cu]Cu-ATSM uptake regions were hypovascular and consisted of tumor cells arrested in the cell cycle, whereas the high [{sup 18}F]FDG uptake regions were hypervascular and consisted of proliferating cells. Conclusion: In our study, it was revealed that one tumor mass contained two regions with different characteristics, which could be distinguished by [{sup 64}Cu]Cu-ATSM and [{sup 18}F]FDG. Because hypoxia and cell cycle arrest are critical factors to reduce tumor sensitivity to radiation and conventional chemotherapy, regions with such characteristics should be treated intensively as one of the primary targets. [{sup 64}Cu]Cu-ATSM, which can delineate hypoxic and cell cycle-arrested regions in tumors, may provide valuable information for cancer treatment as well as possibly for treating such regions directly as an internal radiotherapy reagent.

  2. 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.

  3. 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

  4. 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.

  5. The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5' Flaps and the Removal of 5' Adenylylated Products of Aborted Nick Ligation.

    Science.gov (United States)

    Uson, Maria Loressa; Ghosh, Shreya; Shuman, Stewart

    2017-09-01

    We characterize Mycobacterium smegmatis FenA as a manganese-dependent 5'-flap endonuclease homologous to the 5'-exonuclease of DNA polymerase I. FenA incises a nicked 5' flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3' single strand is eliminated to create a 5'-tailed duplex, FenA incision shifts to between the second and third nucleotides. A double-flap substrate with a mobile junction (mimicking limited strand displacement synthesis during gap repair) is preferentially incised as the 1-nucleotide 3'-flap isomer, with the scissile phosphodiester shifted by one nucleotide versus a static double flap. FenA efficiently removes the 5' App(dN) terminus of an aborted nick ligation reaction intermediate, thereby highlighting FenA as an agent of repair of such lesions, which are formed under a variety of circumstances by bacterial NAD + -dependent DNA ligases and especially by mycobacterial DNA ligases D and C. IMPORTANCE Structure-specific DNA endonucleases are implicated in bacterial DNA replication, repair, and recombination, yet there is scant knowledge of the roster and catalytic repertoire of such nucleases in Mycobacteria This study identifies M. smegmatis FenA as a stand-alone endonuclease homologous to the 5'-exonuclease domain of mycobacterial DNA polymerase 1. FenA incises 5' flaps, 5' nicks, and 5' App(dN) intermediates of aborted nick ligation. The isolated N-terminal domain of M. smegmatis Pol1 is also shown to be a flap endonuclease. Copyright © 2017 American Society for Microbiology.

  6. Konstrukce vřeten vícevřetenového soustružnického automatu

    OpenAIRE

    Kráčmar, Tomáš

    2016-01-01

    Obsahem diplomové práce je konstrukce vřeten vícevřetenového soustružnického automatu pro práci z tyče maximálního průměru 7 mm. Práce se zabývá novou koncepcí pohonu vřeten, kde jsou vřetena poháněna externími asynchronními motory přes ozubená kola s vnitřním ozubením uložená vně vřetenového bubnu namísto současného způsobu pohonu centrálními koaxiálními hřídeli. Součástí práce je rešerše vícevřetenových automatů včetně popisu hlavních uzlů, konstrukční návrh, pevnostní výpočty, výpočty trva...

  7. 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

    2016-01-01

    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. PMID:27557712

  8. On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells.

    OpenAIRE

    Lebon, C.; Rodriguez, G.V.; Zaoui, I.E.; Jaadane, I.; Behar-Cohen, F.; Torriglia, A.

    2015-01-01

    Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess dr...

  9. DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity

    International Nuclear Information System (INIS)

    Gruskin, E.A.; Lloyd, R.S.

    1986-01-01

    T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed

  10. 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

  11. Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6, and rad9 of Saccharomyces cerevisiae. [nicking

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, L [Rochester Univ., N.Y. (USA). Dept. of Radiation Biology and Biophysics; Rochester Univ., N.Y. (USA). School of Medicine and Dentistry)

    1977-10-01

    The ability to remove ultraviolet-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 uv-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.

  12. Low-level overexpression of p53 promotes warfarin-induced calcification of porcine aortic valve interstitial cells by activating Slug gene transcription.

    Science.gov (United States)

    Gao, Li; Ji, Yue; Lu, Yan; Qiu, Ming; Shen, Yejiao; Wang, Yaqing; Kong, Xiangqing; Shao, Yongfeng; Sheng, Yanhui; Sun, Wei

    2018-03-09

    The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification, and increased slug transcription by p53 is required for p53-mediated pAVIC calcification. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. 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.

  14. Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

    Science.gov (United States)

    Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

    2014-09-02

    A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

  15. 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.

  16. Once a physicist: Nick Horvath

    Science.gov (United States)

    Horvath, Nick

    2009-05-01

    How did you get into basketball? That's an easy one: I was tall. I had zero interest in sport at a young age and was pushed into basketball against my will when I was about eight. I hated it. The next year, however, I was so much taller than everyone else that in my first game, even though I had no idea what I was doing, I could simply hold the ball above my head and shoot over everyone. I fell in love with basketball after that first game. If I had played badly that day, it is likely that I would not have played again in my entire life.

  17. C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

    Science.gov (United States)

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

    2009-10-30

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

  18. C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

    Science.gov (United States)

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

    2009-01-01

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

  19. Fisetin and polymeric micelles encapsulating fisetin exhibit potent cytotoxic effects towards ovarian cancer cells.

    Science.gov (United States)

    Xiao, Xue; Zou, Juan; Fang, Yin; Meng, Yibo; Xiao, Chao; Fu, Jiaxin; Liu, Shiyu; Bai, Peng; Yao, Yuan

    2018-03-15

    The anti-tumor activities of Natural compounds and their derivatives are of great interest to pharmaceutical industries. Fisetin is one of prospective natural compounds in this regard but unfortunately with poor hydrophilicity. The effects of unmodified and modified fisetin in cultured ovarian cancer cells were compared by transmission electronmicroscopy to determine apoptotic bodies, MTT assay to quantitate cell numbers, and fluorescence activated cell sorting analyse of various markers to determine the apoptotic state. In addition, the efficacy of fisetin and fisetin-micelles in vivo was determined by using immunocompromised mice. Apoptosis was measured by established markers using both western blot analysis and immunochemistry. Angiogenesis in a xenograft mouse model carring SKOV3 cells was evaluated by color Doppler ultrasound and immunohistochemistry. Multiple lines of evidence indicated that fisetin and fisetin micelles induce apoptosis in ovarian cancer cells in a dose-dependent manner. Histological analysis, terminal deoxynucleotidyltransferase-mediated nick-end labeling assay, western blot, immunohistochemical detection and microvessel density detection demonstrated that fisetin and fisetin micelles induced increased tumor apoptosis, proliferation suppression and antiangiogenesis activities. As far as we know, the present study is the first time to demonstrate the potency of both fisetin and fisetin micelles inducing apoptosis in ovarian cancer cells. Further studies will be needed to validate the therapeutic potential of fisetin and fisetin micelles in ovarian cancer treatment.

  20. The Portraiture of Nick: Scene Two, the Middle Years

    Science.gov (United States)

    Bicehouse, Vaughn L.

    2012-01-01

    In 2007, the Autism Society of America (ASA) estimated that 1.5 million Americans and their families were affected by autism. As the current Autistic Spectrum Disorder (ASD) prevalence rates continue to rise (10%-17% each year) so does the awareness that ASD is no respecter of persons. ASD touches children of every racial, ethnic, and…

  1. 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

  2. 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

  3. 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...... declaratively express timed provisions and obligations as causal relationships between events, and DCR states explicitly represent pending obligations. As key technical results, we show that enforceability of DCR policies is decidable, we give a sufficient polynomial time verifiable condition for a policy...... to be enforceable, and we give an algorithm for determining from a DCR state a sequence of actions that discharge impending obligations....

  4. Review Article: The Vanished Ghosts in Two Hungarian Family Memoirs. Farkas, Charles. 2013. Vanished by the Danube: Peace, War, Revolution, and Flight to the West (Introduction by Margaret McMullan. Albany: Excelsior Editions, State University of New York Press. 472 pp. Illus; and Barlay, Nick. 2013. Scattered Ghosts: One Family's Survival through War, Holocaust and Revolution. London and New York: I. B. Tauris. 240 pp. Illus.

    Directory of Open Access Journals (Sweden)

    Ruth G. Biro

    2015-01-01

    Full Text Available Recent personal documentary works about major historical events of the twentieth century, e.g., World War II, the Holocaust and the 1956 Hungarian Revolution, offer their readers a rich and multifaceted narrative, or a history that is also "his story," "her story" and that of entire families, cohorts and communities. Often, these works are accompanied by visual artifacts such as photographs, family tress, maps etc., or supported by concise historical surveys. Thus these memoirs complete the work of historians with the lived experiences of the few that represent many. Such is the case with two 2013 books by Charles Farkas and Nick Barlay depicting their mid-twentieth century Hungarian families, one Christian and one Jewish, through two World Wars and the anti-communist uprising, culminating in their escape to the West and in the two authors looking back upon the Hungarian past of their families.

  5. Anti-Inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase-3-dependent neutrophil programmed cell death and inhibits NF-kappaB signaling and CXCL8 transcription.

    Science.gov (United States)

    Fischer, Carrie D; Beatty, Jennifer K; Zvaigzne, Cheryl G; Morck, Douglas W; Lucas, Merlyn J; Buret, A G

    2011-01-01

    Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 10(7) CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B(4) in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which

  6. Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.

    OpenAIRE

    Weiss, R S; Lee, S S; Prasad, B V; Javier, R T

    1997-01-01

    An essential oncogenic determinant of subgroup D human adenovirus type 9 (Ad9), which uniquely elicits estrogen-dependent mammary tumors in rats, is encoded by early region 4 open reading frame 1 (E4 ORF1). Whereas Ad9 E4 ORF1 efficiently induces transformed foci on the established rat embryo fibroblast cell line CREF, the related subgroup A Ad12 and subgroup C Ad5 E4 ORF1s do not (R. T. Javier, J. Virol. 68:3917-3924, 1994). In this study, we found that the lack of transforming activity asso...

  7. A Mitochondria-Dependent Pathway Mediates the Apoptosis of GSE-Induced Yeast

    OpenAIRE

    Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun

    2012-01-01

    Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) ...

  8. Paradoxical action of fulvestrant in estradiol-induced regression of tamoxifen-stimulated breast cancer.

    Science.gov (United States)

    Osipo, Clodia; Gajdos, Csaba; Liu, Hong; Chen, Bin; Jordan, V Craig

    2003-11-05

    Long-term tamoxifen treatment of breast cancer can result in tamoxifen-stimulated breast cancer, in which estrogen inhibits tumor growth after tamoxifen withdrawal. We investigated the molecular mechanism(s) of estradiol-induced tumor regression by using an in vivo model of tamoxifen-stimulated human breast cancer. Growth of parental estradiol-stimulated MCF-7E2 and long-term tamoxifen-stimulated MCF-7TAMLT xenografts in athymic mice was measured during treatment with vehicle, estradiol, estradiol plus tamoxifen, tamoxifen alone, estradiol plus fulvestrant, or fulvestrant alone. Apoptosis was detected by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Protein expression was assessed by western blot analysis. mRNA expression was assessed by real-time reverse transcription-polymerase chain reaction. All statistical tests were two-sided. MCF-7E2 tumor growth was stimulated by estradiol (cross-sectional area at week 13 = 1.06 cm2, 95% confidence interval [CI] = 0.82 to 1.30 cm2; Pestradiol-induced regression to 0.18 cm2 (95% CI = 0.15 to 0.21 cm2; P<.001), and tamoxifen or estradiol plus fulvestrant enhanced tumor growth to 1.00 cm2 (95% CI = 0.88 to 1.22 cm2). Estradiol increased the number of apoptotic cells in tumors by 23% (95% CI = 20% to 26%; P<.001) compared with all other treatments, decreased estrogen receptor alpha(ERalpha) protein expression, increased the expression of Fas mRNA and protein, decreased the expression of HER2/neu mRNA and protein and nuclear factor kappaB (NF-kappaB) protein but did not affect Fas ligand protein expression compared with control. Paradoxically, fulvestrant reversed this effect and stimulated MCF-7TAMLT tumor growth apparently through ERalpha-mediated regulation of Fas, HER2/neu, and NF-kappaB. Physiologic levels of estradiol induced regression of tamoxifen-stimulated breast cancer tumors, apparently by inducing the death receptor Fas and suppressing the antiapoptotic

  9. Fatty acid synthase inhibition triggers apoptosis during S phase in human cancer cells.

    Science.gov (United States)

    Zhou, Weibo; Simpson, P Jeanette; McFadden, Jill M; Townsend, Craig A; Medghalchi, Susan M; Vadlamudi, Aravinda; Pinn, Michael L; Ronnett, Gabriele V; Kuhajda, Francis P

    2003-11-01

    C75, an inhibitor of fatty acid synthase (FAS), induces apoptosis in cultured human cancer cells. Its proposed mechanism of action linked high levels of malonyl-CoA after FAS inhibition to potential downstream effects including inhibition of carnitine palmitoyltransferase-1 (CPT-1) with resultant inhibition of fatty acid oxidation. Recent data has shown that C75 directly stimulates CPT-1 increasing fatty acid oxidation in MCF-7 human breast cancer cells despite inhibitory concentrations of malonyl-CoA. In light of these findings, we have studied fatty acid metabolism in MCF7 human breast cancer cells to elucidate the mechanism of action of C75. We now report that: (a) in the setting of increased fatty acid oxidation, C75 inhibits fatty acid synthesis; (b) C273, a reduced form of C75, is unable to inhibit fatty acid synthesis and is nontoxic to MCF7 cells; (c) C75 and 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase, both cause a significant reduction of fatty acid incorporation into phosphatidylcholine, the major membrane phospholipid, within 2 h; (d) pulse chase studies with [(14)C]acetate labeling of membrane lipids show that both C75 and TOFA accelerate the decay of (14)C-labeled lipid from membranes within 2 h; (e) C75 also promotes a 2-3-fold increase in oxidation of membrane lipids within 2 h; and (f) because interference with phospholipid synthesis during S phase is known to trigger apoptosis in cycling cells, we performed double-labeled terminal deoxynucleotidyltransferase-mediated nick end labeling and BrdUrd analysis with both TOFA and C75. C75 triggered apoptosis during S phase, whereas TOFA did not. Moreover, application of TOFA 2 h before C75 blocked the C75 induced apoptosis, whereas etomoxir did not. Taken together these data indicate that FAS inhibition and its downstream inhibition of phospholipid production is a necessary part of the mechanism of action of C75. CPT-1 stimulation does not likely play a role in the

  10. 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. (Curtin U.); (Sydney); (UNC)

    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.

  11. "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".

  12. 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...

  13. Nick Jonas on Type 1 Diabetes | NIH MedlinePlus the Magazine

    Science.gov (United States)

    Skip to main content NIH MedlinePlus the Magazine NIH MedlinePlus Salud Download the Current Issue PDF [1.9 mb] Trusted Health Information from the National Institutes of Health Home Current Issue ...

  14. Triplex configuration in the nick-free DNAs that constitute the chromosomal scaffolds in grasshopper spermatids

    Czech Academy of Sciences Publication Activity Database

    Černá, Adriana; Lopez-Fernandez, C.; Fernandez, J.L.; de la Espina, S.M.D.; De la Torre, C.; Gosalvez, J.

    2008-01-01

    Roč. 117, č. 1 (2008), s. 15-24 ISSN 0009-5915 Institutional research plan: CEZ:AV0Z50380511 Keywords : chromatid scaffold * DNA loops * triplex DNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.111, year: 2008

  15. 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

  16. 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.

  17. 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

  18. 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..."

  19. 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

  20. Preparation of short cytosine-modified oligonucleotides by nicking enzyme amplification reaction

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Hocek, Michal

    2012-01-01

    Roč. 48, č. 55 (2012), s. 6921-6923 ISSN 1359-7345 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : cross - coupling reactions * nucleoside triphosphates * functionalized DNA * restriction endonucleases * polymerase incorporation Subject RIV: CC - Organic Chemistry Impact factor: 6.378, year: 2012

  1. First-generation physical map of the Culicoides variipennis (Diptera: Ceratopogonidae) genome.

    Science.gov (United States)

    Nunamaker, R A; Brown, S E; McHolland, L E; Tabachnick, W J; Knudson, D L

    1999-11-01

    Recombinant cosmids labeled with biotin-11-dUTP or digoxigenin by nick translation were used as in situ hybridization probes to metaphase chromosomes of Culicoides variipennis (Coquillett). Paired fluorescent signals were detected on each arm of sister chromatids and were ordered along the 3 chromosomes. Thirty-three unique probes were mapped to the 3 chromosomes of C. variipennis (2n = 6): 7 to chromosome 1, 20 to chromosome 2, and 6 to chromosome 3. This work represents the first stage in generating a physical map of the genome of C. variipennis.

  2. Apoptosis of gut-associated lymphoid tissue in rainbow trout Oncorhynchus mykiss after incubation with Candida albicans and bacterial lipopolysaccharide.

    Science.gov (United States)

    Passantino, L; Ostillio, A; Cianciotta, A; Russo, C; Carrassi, M; Patruno, R; Dhaskali, L; Passantino, G F; Passantino, A

    2011-06-01

    Until now a few studies have been carried out on the gut lymphoid system in fish despite its protective role in the host. Here, we have evaluated the effects of Candida albicans (Ca) and lipopolysaccaride (LPS) on the pyloric and terminal segments of gut in the rainbow trout Oncorhynchus mykiss. In particular, data show that both Ca and LPS are able to cause apoptosis of intestinal lymphoid cells as detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) procedure. These findings suggest a further modality of gut response in fish to environmental antigens.

  3. Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

    DEFF Research Database (Denmark)

    Larsen, L; Tonnesen, M; Ronn, S G

    2007-01-01

    B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone...... by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone...

  4. Effect of ionizing radiation on apoptosis in the cortex of mouse lymph node

    International Nuclear Information System (INIS)

    Chen Dong; Liu Jiamei; Liu Shuzheng

    1999-01-01

    Objective: To study the alteration of apoptosis in the cortex of mouse lymph node following whole body X-irradiation. Methods: The method of TdT-mediated dUTP nick end labelling (TUNEL) was used to detect apoptosis the cortex of mouse lymph node. Results: The sensitivity to high and low dose ionizing radiation was distinct in different area of the cortex. Conclusion: The decrease of apoptotic cells in the inter nodular and deep cortex indicate that low dose radiation may suppress the apoptosis of T lymphocytes and play a role in immune regulation

  5. Distribuce předpon v českém sylabotónickém trocheji

    Czech Academy of Sciences Publication Activity Database

    Plecháč, Petr; Kolár, Robert; Hlaváčová, J.; Merthová, K.

    2017-01-01

    Roč. 78, č. 4 (2017), s. 322-332 ISSN 0037-7031 R&D Projects: GA ČR(CZ) GA14-31160S Institutional support: RVO:68378068 Keywords : metrics and prosody * morphology * corpus linguistics * trochee * prefixes Subject RIV: AJ - Letters, Mass-media, Audiovision OBOR OECD: Specific literatures Impact factor: 0.625, year: 2016

  6. Fluorescence Quenching in Oligonucleotides Containing 7-Substituted 7-Deazaguanine Bases Prepared by the Nicking Enzyme Amplification Reaction

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Dziuba, Dmytro; Güixens-Gallardo, Pedro; Jurkiewicz, Piotr; Hof, Martin; Hocek, Michal

    2015-01-01

    Roč. 26, č. 2 (2015), s. 361-366 ISSN 1043-1802 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 ; RVO:61388955 Keywords : ethidium bromide * electrochemical detection * DNA Subject RIV: CC - Organic Chemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 4.500, year: 2015

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

    International Nuclear Information System (INIS)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

    2015-01-01

    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

  8. 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.

  9. 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..."

  10. In the Nick of Time: A Heteroskedastic SVAR Model and Its Application to the Crude Oil Futures Market

    NARCIS (Netherlands)

    Sun, Hang; Bos, Jaap W.B.; Li, Zhuo

    2017-01-01

    Many economic analyses revolve around the identification of shocks. However, this becomes difficult if we do not have enough information, for example because we do not observe the underlying process at a high enough frequency. As a result, if the response of one variable to a shock to another takes

  11. 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

  12. 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..."

  13. Programmed cell death of tobacco BY-2 cells induced by still culture conditions is affected by the age of the culture under agitation.

    Science.gov (United States)

    Hiraga, Asahi; Kaneta, Tsuyoshi; Sato, Yasushi; Sato, Seiichi

    2010-01-25

    Evans Blue staining indicated that actively growing tobacco BY-2 cells in the exponential phase died more rapidly than quiescent cells in the stationary phase when the cells cultured under agitation were placed under still conditions. Fifty percent cell death was induced at about 18, 26, 80 and 140 h for early, mid, late exponential- and stationary-phase cells, respectively. Actively growing cells became TUNEL (transferase-mediated dUTP nick end labelling)-positive more rapidly than quiescent cells, suggesting that the cell death evaluated by Evans Blue is accompanied by DNA cleavages. Electrophoresis of genomic DNA showed a typical 'DNA laddering' pattern formed by multiples of about 200 bp internucleosomal units. Chromatin condensation was first detected at least within 24 h by light microscopy, and then cell shrinkage followed. These findings suggest that the death of BY-2 cells induced by still conditions is PCD (programmed cell death).

  14. Population control of resident and immigrant microglia by mitosis and apoptosis

    DEFF Research Database (Denmark)

    Wirenfeldt, Martin; Dissing-Olesen, Lasse; Babcock, Alicia

    2007-01-01

    microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate...... in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced...... in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations....

  15. Study of influence of catechins on bystander responses in alpha-particle radiobiological experiments using thin PADC films

    International Nuclear Information System (INIS)

    Law, Y.L.; Yu, K.N.

    2009-01-01

    In this study, Chinese hamster ovary (CHO) cells were cultured in custom-made petri dishes with thin PADC films as substrates. Alpha particles with energies of 5 MeV were then irradiated from the bottom of PADC films. The DNA strand breaks in the bystander cells induced by irradiation were quantified with the use of terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay. To study the influence of catechins on the bystander responses, catechins were added into the medium before alpha-particle irradiation of the cells. Fewer DNA strand breaks in the bystander cells were observed. As catechins are ROS (reactive oxygen species)-scavengers, the studied bystander cells might have been protected from radiation through scavenging of ROS by catechins.

  16. Study of influence of catechins on bystander responses in alpha-particle radiobiological experiments using thin PADC films

    Energy Technology Data Exchange (ETDEWEB)

    Law, Y.L. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Yu, K.N., E-mail: peter.yu@cityu.edu.h [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong)

    2009-10-15

    In this study, Chinese hamster ovary (CHO) cells were cultured in custom-made petri dishes with thin PADC films as substrates. Alpha particles with energies of 5 MeV were then irradiated from the bottom of PADC films. The DNA strand breaks in the bystander cells induced by irradiation were quantified with the use of terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay. To study the influence of catechins on the bystander responses, catechins were added into the medium before alpha-particle irradiation of the cells. Fewer DNA strand breaks in the bystander cells were observed. As catechins are ROS (reactive oxygen species)-scavengers, the studied bystander cells might have been protected from radiation through scavenging of ROS by catechins.

  17. Evaluation of Dying Vocal Fold Epithelial Cells by Ultrastructural Features and TUNEL Method

    Science.gov (United States)

    Novaleski, Carolyn K.; Mizuta, Masanobu; Rousseau, Bernard

    2016-01-01

    Cell death is a regulated mechanism of eliminating cells to maintain tissue homeostasis. This study described two methodological procedures for evaluating cell death in the epithelium of immobilized, approximated, and vibrated vocal folds from 12 New Zealand white breeder rabbits. The gold standard technique of transmission electron microscopy evaluated high-quality ultrastructural criteria of cell death and a common immunohistochemical marker, terminal deoxynucleotidyl transferase dUTP nick end labeling method, to confirm cell death signaling. Results revealed that ultrastructural characteristics of apoptotic cell death, specifically condensed chromatin and apoptotic bodies, were observed after vocal fold vibration and approximation. Although episodes of necrotic cell death were rare, few enlarged cell nuclei were present after vibration and approximation. The vocal fold expresses an immunohistochemical marker for apoptosis along the apical surface of the epithelium. This study provides a solid foundation for future investigations regarding the role of cell death in vocal fold health and disease. PMID:27537846

  18. Detection of programmed cell death in plant embryos.

    Science.gov (United States)

    Filonova, Lada H; Suárez, María F; Bozhkov, Peter V

    2008-01-01

    Programmed cell death (PCD) is an integral part of embryogenesis. In plant embryos, PCD functions during terminal differentiation and elimination of the temporary organ, suspensor, as well as during establishment of provascular system. Embryo abortion is another example of embryonic PCD activated at pathological situations and in polyembryonic seeds. Recent studies identified the sequence of cytological events leading to cellular self-destruction in plant embryos. As in most if not all the developmental cell deaths in plants, embryonic PCD is hallmarked by autophagic degradation of the cytoplasm and nuclear disassembly that includes breakdown of the nuclear envelope and DNA fragmentation. The optimized setup of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) allows the routine in situ analysis of nuclear DNA fragmentation in plant embryos. This chapter provides step-by-step procedure of how to process embryos for TUNEL and how to combine TUNEL with immunolocalization of the protein of interest.

  19. Effect of ionizing radiation on apoptosis in mouse Peyer's patches

    International Nuclear Information System (INIS)

    Liu Jiamei; Chen Dong; Liu Shuzheng

    1999-01-01

    The relationship of time-effect and dose-effect of apoptosis in mouse Peyer's patches after whole body irradiation (WBI) with different doses of X-rays was studied by the method of TdT-mediated dUTP nick end labelling (TUNEL). The results showed that the number of TUNEL positive cells in mouse Peyer's patches were significantly increased following WBI with 2 Gy irradiation, While the number of TUNEL positive cells were decreased after WBI with doses of 0.05 Gy and 0.075 Gy X-rays. the results support the view that 2 Gy irradiation promote the apoptosis of immune cells and the low doses of radiation suppress the apoptosis of immune cells

  20. 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.

  1. Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells

    International Nuclear Information System (INIS)

    Mizobuchi, Masahide; Ogata, Hiroaki; Hatamura, Ikuji; Saji, Fumie; Koiwa, Fumihiko; Kinugasa, Eriko; Koshikawa, Shozo; Akizawa, Tadao

    2007-01-01

    Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10 -4 M), or 10% cyclodextrin, for 6 h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro

  2. Hyperoxygenated hydrogen-rich solution suppresses shock- and resuscitation-induced liver injury.

    Science.gov (United States)

    Dang, Yangjie; Liu, Ting; Mei, Xiaopeng; Meng, Xiangzhong; Gou, Xingchun; Deng, Bin; Xu, Hao; Xu, Lixian

    2017-12-01

    It is not known whether simultaneous delivery of hydrogen and oxygen can reduce injury caused by hemorrhagic shock and resuscitation (HSR). This study investigated the therapeutic potential of hyperoxygenated hydrogen-rich solution (HHOS), a combined hydrogen/oxygen carrier, in a rat model of HSR-induced liver injury. Rats (n = 60) were randomly divided into 5 groups (n = 6 per group at each time point). One group underwent sham operation, and the others were subjected to severe hemorrhagic shock and then treated with lactated Ringer's solution (LRS), hydrogen-rich solution, hyperoxygenated solution, or HHOS. At 2 and 6 h after resuscitation, blood samples (n = 6) were collected from the femoral artery and serum concentrations of alanine aminotransferase and aspartate aminotransferase (AST) were measured. Rats were then sacrificed, and histopathological changes in the liver were evaluated by quantifying the percentage of apoptotic cells by caspase-3 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick-end labeling. Inflammation was assessed by assessing malondialdehyde content and tumor necrosis factor-α, and interleukin (IL)-6 expression. Compared to lactated Ringer's solution, hydrogen-rich solution, or hyperoxygenated solution groups, serum AST and alanine aminotransferase levels and IL-6, tumor necrosis factor-α, and malondialdehyde expression in liver tissue were decreased by HHOS treatment. The number of caspase-3- and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells was decreased (P < 0.05) by HHOS treatment, 2 and 6 h after resuscitation. HHOS has protective effects against liver injury in a rat model of HSR. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

    Science.gov (United States)

    Lavazza, Cristiana; Carlo-Stella, Carmelo; Giacomini, Arianna; Cleris, Loredana; Righi, Marco; Sia, Daniela; Di Nicola, Massimo; Magni, Michele; Longoni, Paolo; Milanesi, Marco; Francolini, Maura; Gloghini, Annunziata; Carbone, Antonino; Formelli, Franca; Gianni, Alessandro M

    2010-03-18

    Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

  4. Nickel(I) and nickel(III) complexes of substituted tetraaza macrocycles formed by pulse radiolysis and electrochemistry of nickel(II) precursors

    International Nuclear Information System (INIS)

    Bernhardt, P.V.; Lawrance, G.A.; Sangster, D.F.

    1988-01-01

    The square-planar nickel(II) complexes of the ligands 8-methyl-8-nitro-1,3,6,10,13,15-hexaazatricyclo[13.1.1.1/sup 13,15/]octadecane, 8-amino-8-methyl-1,3,6,10,13,15-hexaazatricyclo[13.1.1.1/sup 13,15/]octadecane, 3,7-bis(2-aminoethyl)-1,3,5,7-tetraazabicyclo[3.3.1]nonane, and 9-methyl-9-nitro-1,4,7,11-tetraazacyclotridecane (I-IV) react rapidly with hydroxyl radicals and aquated electrons (e/sub aq/). The initial transient products of these reactions decay via first-order kinetics within a few milliseconds in neutral aqueous solution at 22/degrees/C in all cases. Electronic spectra and decay rate constants, as well as formation rate constants, are reported for all transients. Reaction of the nitro-substituted complexes with e/sub aq/ led to electron addition to the nitro group rather than to the metal center; otherwise, a Ni/sup I/ transient is observed. Following reaction with OH, the product of the initial decay remains a Ni/sup III/ species. This is more long-lived, and stabilization of Ni/sup III/ by axial coordination of the pendant amine in II is indicated. No notable stabilization of Ni/sup I/ or Ni/sup III/ from the presence of the bicyclic azamethylene football in I-III occurs. Cyclic voltammetry in acetonitrile identified both one-electron oxidation and one-electron reduction processes for the nickel(II) complexes, as well as nitro group reduction, where this group was pendant to the macrocycle. 34 references, 3 figures, 3 tables

  5. Assaying Break and Nick-Induced Homologous Recombination in Mammalian Cells Using the DR-GFP Reporter and Cas9 Nucleases

    NARCIS (Netherlands)

    Vriend, Lianne E. M.; Jasin, Maria; Krawczyk, Przemek M.

    2014-01-01

    Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the

  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. Srovnání organizačních struktur vybraných mládežnických klubů

    OpenAIRE

    Vavříček, Jaroslav

    2006-01-01

    and keywords Title: Comparison of organizational structures of selected youth clubs The Goals: Specify the terms connected with the thesis, work out the methodology part, introduce selected clubs and their organizational structures, interview members and employees ofthese dubs, present my idea. Methodology: Organizational structures of selected soccer dubs and the ways oftheir funding. Suggestion of an idea! organizational structure, informations also acquirred by nonstandard interviews with ...

  8. 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..."

  9. An energy source which has come just in the nick of time; Une source d'energie qui tombe pile

    Energy Technology Data Exchange (ETDEWEB)

    Jacquemin, Ch.

    1999-12-01

    Fifty-six projects from industrialists and public research organisms have been proposed to the 'fuel cell' research and technological innovation network. Moreover, a project carried out by the French PSA/Renault car manufacturers and devoted to the development of vehicles equipped with fuel cells has been labelled by the network. This short paper summarizes the aim and organization of the fuel cell network in the French scientific and technical program. (J.S.)

  10. 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.

  11. 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..."

  12. Vorliegende Gedichte stammen aus dem Zyklus "Der Handgänger" : [luuletused] / Kristiina Ehin ; eesti keelest tlk Gisbert Jänicke ja Irja Grönholm

    Index Scriptorium Estoniae

    Ehin, Kristiina, 1977-

    2006-01-01

    Luuletusi tsüklist "Kätelkäija" (Kaitseala. Tallinn : Huma, 2005). - Andmeid autori kohta lk. 61. Sisu: Der Handgänger = Kätelkäija : "draussen ist üppiger juli..." = "väljas on lopsakas juuli..." ; "ich menschliche hülle keuche über die erde..." = "ihulise inimesena hingeldan mööda maad..." ; "die einsamen waldseen..." = "üksildased metsajärved..." ; "im schüttelfrost kam sie ans ufer..." = "kaldalejõudmine külmavärinais..."

  13. 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.

  14. Nářeční lexikum na východní Moravě a karpatská salašnická kolonizace

    Czech Academy of Sciences Publication Activity Database

    Goláňová, Hana

    -, č. 4 (2016), s. 316-327 ISSN 0862-8351 R&D Projects: GA ČR(CZ) GA16-04648S Institutional support: RVO:68378092 Keywords : Carpathian shepherd colonization * Obščekarpatskij dialektologičeskij atlas * dialectal lexicon * carpathism * eastern-Moravian dialect Subject RIV: AI - Linguistics

  15. Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Raindlová, Veronika; Hocek, Michal

    2013-01-01

    Roč. 24, č. 6 (2013), s. 1081-1093 ISSN 1043-1802 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : isothermal DNA amplification * cross - coupling reactions * nucleoside triphosphates * polymerase incorporation * functionalized DNA * nucleic-acids Subject RIV: CC - Organic Chemistry Impact factor: 4.821, year: 2013

  16. Effect of Transient Maternal Hypotension on Apoptotic Cell Death in Foetal Rat Brain

    Directory of Open Access Journals (Sweden)

    Hamit Özyürek

    2014-03-01

    Full Text Available Background: Intrauterine perfusion insufficiency induced by transient maternal hypotension has been reported to be associated with foetal brain malformations. However, the effects of maternal hypotension on apoptotic processes in the foetal brain have not been investigated experimentally during the intrauterine period. Aims: The aim of this study was to investigate the effects of transient maternal hypotension on apoptotic cell death in the intrauterine foetal brain. Study Design: Animal experimentation. Methods: Three-month-old female Wistar albino rats were allocated into four groups (n=5 each. The impact of hypoxic/ischemic injury induced by transient maternal hypotension on the 15th day of pregnancy (late gestation in rats was investigated at 48 (H17 group or 96 hours (H19 group after the insult. Control groups underwent the same procedure except for induction of hypotension (C17 and H17 groups. Brain sections of one randomly selected foetus from each pregnant rat were histopathologically evaluated for hypoxic/ischemic injury in the metencephalon, diencephalon, and telencephalon by terminal transferase-mediated dUTP nick end labelling and active cysteine-dependent aspartate-directed protease-3 (caspase-3 positivity for cell death. Results: The number of terminal transferase-mediated dUTP nick end labelling (+ cells in all the areas examined was comparable in both hypotension and control groups. The H17 group had active caspase-3 (+ cells in the metencephalon and telencephalon, sparing diencephalon, whereas the C19 and H19 groups had active caspase-3 (+ cells in all three regions. The number of active caspase-3 (+ cells in the telencephalon in the H19 group was higher compared with the metencephalon and diencephalon and compared with H17 group (p<0.05. Conclusion: Our results suggest that prenatal hypoxic/ischemic injury triggers apoptotic mechanisms. Therefore, blockade of apoptotic pathways, considering the time pattern of the insult, may

  17. 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.

  18. Bacterial reduction in genotoxicity of Direct Red 28 dye.

    Science.gov (United States)

    Bafana, Amit; Jain, Minakshi; Agrawal, Gaurav; Chakrabarti, Tapan

    2009-03-01

    Direct Red 28 (DR28) is a benzidine-based azo dye widely used in several countries. It has also been a subject of intense research for its anti-prion activity. Like other benzidine-based azo dyes, it is also carcinogenic and toxic. However, there are very few studies addressing its detoxification. In the present study, a Bacillus velezensis strain was used for detoxification of DR28. Toxicity was checked by a battery of highly sensitive genotoxicity assays like comet assay, DNA ladder formation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and flow cytometric Annexin V binding assay. HL-60 cell line was used as the test system. All the assays showed an initial increase in toxicity upon biodegradation due to release of mutagenic products, like benzidine and 4-aminobiphenyl, from the dye. These intermediates caused significant DNA damage and induced apoptosis in HL-60 cells. Then the culture degraded these mutagenic intermediates, due to which the toxicity was reduced gradually, finally resulting in nearly complete detoxification.

  19. Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine.

    Science.gov (United States)

    Yeung, L Y; Wai, Maria S M; Fan, Ming; Mak, Y T; Lam, W P; Li, Zhen; Lu, Gang; Yew, David T

    2010-03-15

    Ketamine, a non-competitive antagonist at the glutamatergic N-methyl-d-aspartate (NMDA) receptor, might impair memory function of the brain. Loss of memory is also a characteristic of aging and Alzheimer's disease. Hyperphosphorylation of tau is an early event in the aging process and Alzheimer's disease. Therefore, we aimed to find out whether long-term ketmaine administration is related to hyperphosphorylation of tau or not in the brains of mice and monkeys. Results showed that after 6 months' administration of ketamine, in the prefrontal and entorhinal cortical sections of mouse and monkey brains, there were significant increases of positive sites for the hyperphosphorylated tau protein as compared to the control animals receiving no ketamine administration. Furthermore, about 15% of hyperphosphorylated tau positive cells were also positively labeled by terminal dUTP nick end labeling (TUNEL) indicating there might be a relationship between hyperphosphorylation of tau and apoptosis. Therefore, the long-term ketamine toxicity might involve neurodegenerative process similar to that of aging and/or Alzheimer's disease. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  20. 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 % .

  1. 3-Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines.

    Science.gov (United States)

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Kwak, Byung Kook; Loffroy, Romaric; Vali, Mustafa

    2010-03-01

    Autophagy, a cellular response to stress, plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). Recently, we reported that the pyruvate analog 3-bromopyruvate (3-BrPA) promoted tumor cell death by targeting GAPDH. In continuance, we investigated the intracellular response of two human HCC cell lines (Hep3B and SK-Hep1) that differ in their status of key apoptotic regulators, p53 and Fas. 3-BrPA treatment induced endoplasmic reticulum (ER) stress, translation inhibition and apoptosis based on Western blot and qPCR, pulse labeling, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 in both the cell lines. However, electron microscopy revealed that 3-BrPA treated SK-Hep1 cells underwent classical apoptotic cell death while Hep3B cells initially responded with the protective autophagy that failed to prevent eventual apoptosis. 3-BrPA treatment promotes apoptosis in human HCC cell lines, irrespective of the intracellular response.

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

    Science.gov (United States)

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

    2013-08-15

    From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12(th) day of pregnancy, 300 mg/kg rine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neonatal 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 cells in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cell apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. nohistochemical staining revealed that taurine supplement increased glial cell line-derived neurotrophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cell apoptosis through the glial cell line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.

  3. A high ratio of apoptosis to proliferation correlates with improved survival after radiotherapy for cervical adenocarcinoma

    International Nuclear Information System (INIS)

    Sheridan, Mary T.; Cooper, Rachel A.; West, Catharine M.L.

    1999-01-01

    Purpose: A retrospective study was made of the role of apoptosis in determining radiotherapy outcome in 39 adenocarcinoma of the cervix. A comparison was also made of the detection of apoptosis by morphology and the TdT dUtp nick end-labeling (TUNEL) assay. Methods and Materials: The level of apoptosis was assessed in paraffin-embedded sections by cell morphology, the TUNEL assay, and a combination of the two. A total of 2,000 cells were counted per section, to obtain apoptotic (AI) and mitotic (MI) indices. Results: Patients with a high AI had a higher survival rate than those with a low AI, however, the difference was not significant. Using a ratio of apoptosis to proliferation indices, patients with an AI:MI > median had significantly better survival than those with AI:MI < median. This was true where the AI was quantified by morphology alone (p = 0.030) or in combination with the TUNEL assay (p = 0.008). Where the AI was quantified by a combination of morphology and TUNEL, the 5-year survival rates for women with AI:MI greater or less than the median were 81% and 25%, respectively. Conclusion: A high ratio of AI:MI in adenocarcinoma of the cervix indicates a good prognosis. A combination of the TUNEL assay and morphology provided the best discrimination between outcome groups

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

    International Nuclear Information System (INIS)

    Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei; Tzeng, Wen-Pei; Hsiao, Che-Jen; Liu, Shih-Chia; Tang, Chih-Hsin

    2013-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Edward Rockenstein

    2015-01-01

    Full Text Available 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.

  8. Effect of erythropoietin on acoustically traumatized rat cochlea: an immunohistochemical study.

    Science.gov (United States)

    Gürgen, Oğuzhan; Gürgen, Seren Gülşen; Kirkim, Günay; Kolatan, Efsun; Gürkan, Selhan; Güvenç, Yeşim; Eskiizmir, Görkem

    2014-08-01

    To investigate the audiological and histopathological effects of erythropoietin on acoustic overstimulation in rats. Twenty-two male Wistar albino rats were divided into 3 groups: sham group (n = 7), erythropoietin injection group (n = 8), and saline injection group (n = 7). Both erythropoietin and saline injection groups were exposed to white noise (100 decibel [dB] sound pressure level [SPL]) for 3 hours. Auditory brainstem responses were measured before, immediately after, and on the 7th day of noise exposure. All animals were sacrificed on the 7th day and temporal bones were collected. The serial sections of the cochleae were stained by caspase-3 and caspase-9 immunostaining and by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method in order to detect apoptotic cells. In the saline group statistically significant differences were detected between the baseline and immediate postacoustic overstimulation thresholds of click and 6 kHz stimuli. However, when the baseline and immediate postacoustic overstimulation thresholds of click and 6 kHz stimuli were compared in the erythropoietin injection group, no statistically significant difference was determined. Histopathologic evaluations demonstrated that erythropoietin decreased the amount of apoptotic cells in the cochlea. Erythropoietin is likely to prevent the acute threshold changes and decrease the amount of apoptosis in cochlea after acoustic overstimulation in rats.

  9. Time-specific blockade of PDGFR with Imatinib (Glivec®) causes cataract and disruption of lens fiber cells in neonatal mice.

    Science.gov (United States)

    Zhou, Yin-Pin; He, Yang-Tao; Chen, Cheng-Li; Ji, Jun; Niu, Jian-Qin; Wang, Han-Zhi; Li, Shi-Feng; Huang, Lan; Mei, Feng

    2011-03-01

    This study aimed at investigating the response of lens epithelial cells in postnatal mice to Imatinib (Glivec®, a potent inhibitor of platelet-derived growth factor receptor (PDGFR)) treatment. Mouse eyes were sampled 10 days after administration of Imatinib (0.5 mg·g(-1)·day(-1)) for 3 days, at either 7, 14, or 21 days postpartum. Structural changes of lens were revealed by routine H.E. staining. Levels of proliferation and apoptosis were revealed by BrdU incorporation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively, and immunofluorescent staining with anti-PDGFRα antibody was carried out on the sections of eyeball. PDGFRα and p-PDGFRαprotein levels were evaluated by Western blot. Our results indicated that administration of Imatinib led to blockade of PDGFR signaling. Formation of cataracts was found only in those mice where treatment started from 7 days postpartum (P7), but was not observed in those samples from P14 nor P21. Fiber cells were disorganized in cataract lens core as observed histologically, and migration of epithelial cells was also inhibited. No apoptosis was detected with the TUNEL method. Our results indicated blockade of PDGFR at the neonatal stage (P7) would lead to cataracts and lens fiber cells disorganization, suggesting that PDGFR signaling plays a time-specific and crucial role in the postnatal development of lens in the mouse, and also may provide a new approach to produce a congenital cataract animal model.

  10. 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.

  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. Inhibitory effects of CP on the growth of human gastric adenocarcinoma BGC-823 tumours in nude mice.

    Science.gov (United States)

    Wang, Hai-Jun; Liu, Yu; Zhou, Bao-Jun; Zhang, Zhan-Xue; Li, Ai-Ying; An, Ran; Yue, Bin; Fan, Li-Qiao; Li, Yong

    2018-05-01

    Objective To investigate the potential antitumour effects of [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonic acid (CP) against gastric adenocarcinoma. Methods Human BGC-823 xenotransplants were established in nude mice. Animals were randomly divided into control and CP groups, which were administered NaHCO 3 vehicle alone or CP dissolved in NaHCO 3 (200 µg/kg body weight) daily, respectively. Tumour volume was measured weekly for 6 weeks. Resected tumours were assayed for proliferative activity with anti-Ki-67 or anti-proliferating cell nuclear antigen (PCNA) antibodies. Cell apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays and with caspase-3 immunostaining. Proteins were measured by Western blotting. Results There was a significant reduction in tumour volume and a reduced percentage of Ki-67-positive or PCNA-positive cells in the CP group compared with the control group. The percentage of TUNEL-positive or caspase 3-positive cells significantly increased following CP treatment compared with the control group. Tumours from the CP group had higher levels of phosphorylated-extracellular signal-regulated kinase (p-ERK) and phosphorylated-AKT (p-AKT) compared with control tumours. Conclusion CP treatment inhibited tumour growth and induced tumour cell apoptosis in a nude mouse model of BGC-823 gastric adenocarcinoma. Activation of the AKT and ERK signalling pathways may mediate this antitumour activity.

  13. 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

  14. A Cytotoxic Hydroperoxy Sterol from the Brown Alga, Nizamuddinia Zanardinii

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    Abdolhossein Rustaiyan

    2013-03-01

    Full Text Available Background:The marine environment is a unique source of bioactive natural products, of which Nizamuddinia zanardinii is an important brown algae distributed in Oman Sea. Literature revealed that there is no report on phytochemistry and pharmacology of this valuable algae.Methods:Bioguided fractionation of the methanolic extract of Nizamuddinia zanardinii, collected from Oman Sea, led to the isolation of a hydroperoxy sterol. Its structure was determined by analysis of the spectroscopic data as 24-hydroperoxy-24-vinyl cholesterol (HVC. In vitro cytotoxic activity of this compound was evaluated against HT29, MCF7, A549, HepG2 and MDBK cell lines.Results:Although 24(R-hydroproxy-24-vinylcholesterol has been previously reported from Sargassum and Padina species, it is the first report on the presence of this compound from N. zanardinii. This compound exhibited cytotoxicity in all cell lines (IC50, 3.62, 9.09, 17.96, 32.31 and 37.31 μg/mL respectively. HVC was also evaluated for apoptotic activity and demonstrated positive results in terminal deoxynucleotidyl transferase dUTP Nick End labeling (TUNEL assay suggesting it a candidate for further apoptotic studies.Conclusions:Nizamuddinia zanardinii, a remarkable brown algae of Oman Sea, is a good source of hydroproxy sterols with promising cytotoxic on various cell lines particularly human colon adenocarcinoma.

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

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    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.

  16. A mitochondria-dependent pathway mediates the apoptosis of GSE-induced yeast.

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    Sishuo Cao

    Full Text Available Grapefruit seed extract (GSE, which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL and 4,6'-diaminidino-2-phenylindole (DAPI staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential and ROS (reactive oxygen species indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA. The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS. We found that the changes of the metabolites and the protein changes had relevant consistency.

  17. 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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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 H_2O_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_2O_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.

  19. Fluorine-induced apoptosis and lipid peroxidation in human hair follicles in vitro.

    Science.gov (United States)

    Wang, Zheng-hui; Li, Xiao-li; Yang, Zhuang-qun; Xu, Min

    2010-12-01

    Fluoride is an essential trace element for human body; however, exposure to high amounts of fluoride has been documented to be correlated with an increasing risk of hair loss. To date, little is known about the mechanism(s) of how fluoride affects hair follicles. Here, we demonstrated that middle (1.0 mmol/L) and high (10.0 mmol/L) concentrations of sodium fluoride (NaF) significantly inhibited hair follicle elongation in vitro, but low NaF (0.1 mmol/L) showed little influence. Moreover, treatment with high levels of NaF resulted in a marked increase in terminal dUTP nick end labeling-positive cells in the outer layer of the outer root sheath, the dermal sheath, and the lower bulb matrix surrounding dermal papilla. Furthermore, the enhanced apoptosis was coupled with an increased oxidative stress manifested as higher malondialdehyde content. Additionally, the presence of selenium considerably antagonized the effects of middle NaF on hair follicles, with regard to either the suppression of hair growth or the induction of oxidative stress and apoptosis. In conclusion, exposure to high levels of fluoride compromises hair follicle growth and accelerate cell apoptosis in vitro. The toxicity of fluoride can be reduced by selenium, at least partially via the suppression of intracellular oxidative stress.

  20. Stimulatory effects of curcumin and quercetin on posttranslational modifications of p53 during lung carcinogenesis.

    Science.gov (United States)

    Zhang, P; Zhang, Xy

    2018-06-01

    Lung cancer is responsible for increase in mortality due to cancer-related deaths, and new approaches are being explored for the betterment of the situation. In the present study, chemopreventive efficacy of curcumin and quercetin was investigated against benzo(a)pyrene (BP)-induced lung carcinogenesis. The mice were segregated into five groups, which included normal control, BP-treated, BP + curcumin-treated, BP + quercetin-treated, and BP + curcumin + quercetin-treated groups. The morphological and histological analyses of tumor nodules confirmed lung carcinogenesis22 weeks after weeks single intraperitoneal injection of BP at a dose of 100 mg/kg body weight to mice. Curcumin and quercetin when administered individually as well as in combination significantly elevated the expression of acetylated-p53, which was otherwise depressed due to BP treatment. Also, both the phytochemicals significantly reduced the BP-inflicted increased levels of phosphorylated-p53. Furthermore, observed increase in the number of apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), assay and increased activities of caspase 3 and 9 confirmed the induction of apoptosis by curcumin and quercetin. Moreover, the histological slides also showed noticeable improvement in the histoarchitecture of lungs by phytochemicals. The present study concludes that prophylactic treatment with curcumin and quercetin induces apoptosis in the lungs by modulation of p53 posttranslational modifications.

  1. Arsenic induced apoptosis in rat liver following repeated 60 days exposure

    International Nuclear Information System (INIS)

    Bashir, Somia; Sharma, Yukti; Irshad, M.; Nag, T.C.; Tiwari, Monica; Kabra, M.; Dogra, T.D.

    2006-01-01

    Background: Accumulation of the wide spread environmental toxin arsenic in liver results in hepatotoxcity. Exposure to arsenite and other arsenicals has been previously shown to induce apoptosis in certain tumor cell lines at low (1-3 μM) concentration. Aim: The present study was focused to elucidate the role of free radicals in arsenic toxicity and to investigate the nature of in vivo sodium arsenite induced cell death in liver. Methods: Male wistar rats were exposed to arsenite at three different doses of 0.05, 2.5 and 5 mg/l for 60 days. Oxidative stress in liver was measured by estimating pro-oxidant and antioxidant activity in liver. Histopathological examination of liver was carried out by light and transmission electron microscopy. Analysis of DNA fragmentation by gel electrophoresis was used to identify apoptosis after the exposure. Terminal deoxy-nucleotidyl transferase mediated dUTP Nick end-labeling (TUNEL) assay was used to qualify and quantify apoptosis. Results: A significant increase in cytochrome-P450 and lipid peroxidation accompanied with a significant alteration in the activity of many of the antioxidants was observed, all suggestive of arsenic induced oxidative stress. Histopathological examination under light and transmission electron microscope suggested a combination of ongoing necrosis and apoptosis. DNA-TUNEL showed an increase in apoptotic cells in liver. Agarose gel electrophoresis of DNA of hepatocytes resulted in a characteristic ladder pattern. Conclusion: Chronic arsenic administration induces a specific pattern of apoptosis called post-mitotic apoptosis

  2. Detection of radiation-induced apoptosis using the comet assay

    International Nuclear Information System (INIS)

    Wada, Seiichi; Kobayashi, Yasuhiko; Funayama, Tomoo; Yamamoto, Kazuo; Khoa, Tran Van; Natsuhori, Masahiro; Ito, Nobuhiko

    2003-01-01

    The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to satin the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis. (author)

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

    International Nuclear Information System (INIS)

    Xia, D.Y.; Li, W.; Qian, H.R.; Yao, S.; Liu, J.G.; Qi, X.K.

    2013-01-01

    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

  4. Ablation of phosphoinositide 3-kinase-gamma reduces the severity of acute pancreatitis.

    Science.gov (United States)

    Lupia, Enrico; Goffi, Alberto; De Giuli, Paolo; Azzolino, Ornella; Bosco, Ornella; Patrucco, Enrico; Vivaldo, Maria Cristina; Ricca, Marco; Wymann, Matthias P; Hirsch, Emilio; Montrucchio, Giuseppe; Emanuelli, Giorgio

    2004-12-01

    In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-gamma (PI3K gamma) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3K gamma, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3K gamma significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3K gamma-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3K gamma, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3K gamma. Our results thus suggest that inhibition of PI3K gamma may be of therapeutic value in acute pancreatitis.

  5. Ablation of Phosphoinositide 3-Kinase-γ Reduces the Severity of Acute Pancreatitis

    Science.gov (United States)

    Lupia, Enrico; Goffi, Alberto; De Giuli, Paolo; Azzolino, Ornella; Bosco, Ornella; Patrucco, Enrico; Vivaldo, Maria Cristina; Ricca, Marco; Wymann, Matthias P.; Hirsch, Emilio; Montrucchio, Giuseppe; Emanuelli, Giorgio

    2004-01-01

    In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-γ (PI3Kγ) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3Kγ, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3Kγ significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3Kγ-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3Kγ, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3Kγ. Our results thus suggest that inhibition of PI3Kγ may be of therapeutic value in acute pancreatitis. PMID:15579443

  6. Edaravone Protect against Retinal Damage in Streptozotocin-Induced Diabetic Mice

    Science.gov (United States)

    Liu, Xiaoyi; Chen, Xi; Xie, Ping; Yuan, Songtao; Zhang, Weiwei; Lin, Xiaojun; Liu, Qinghuai

    2014-01-01

    Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the clinical treatment of retinal injury. In this study, we investigated the protective effects of edaravone against diabetic retinal damage in the mouse. Diabetic retinopathy in the mouse was induced by injection of streptozotocin. Edaravone was given once-daily and was intraperitoneally (i.p.) treated at a dose of 3 mg/kg from streptozotocin injection to 4 weeks after onset of diabetes. Retinal ganglion cells (RGCs) damage was evaluated by recording the pattern electroretinogram (ERG). RGCs damage was also detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and the levels of reactive oxygen species (ROS) were determined fluorometrically. The expressions of phosporylated-ERK1/2, BDNF, and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expression of BDNF was significantly decreased in the retinas of diabetic mice, compared to nondiabetic mice. Administration of edaravone significantly attenuated diabetes induced RGCs death, upregulation of ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF. These findings suggest that oxidative stress plays a pivotal role in diabetic retinal damage and that systemic administration of edaravone may slow the progression of retinal neuropathy induced by diabetes. PMID:24897298

  7. Edaravone protect against retinal damage in streptozotocin-induced diabetic mice.

    Directory of Open Access Journals (Sweden)

    Dongqing Yuan

    Full Text Available Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, a free radical scavenger, is used for the clinical treatment of retinal injury. In this study, we investigated the protective effects of edaravone against diabetic retinal damage in the mouse. Diabetic retinopathy in the mouse was induced by injection of streptozotocin. Edaravone was given once-daily and was intraperitoneally (i.p. treated at a dose of 3 mg/kg from streptozotocin injection to 4 weeks after onset of diabetes. Retinal ganglion cells (RGCs damage was evaluated by recording the pattern electroretinogram (ERG. RGCs damage was also detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining, and the levels of reactive oxygen species (ROS were determined fluorometrically. The expressions of phosporylated-ERK1/2, BDNF, and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expression of BDNF was significantly decreased in the retinas of diabetic mice, compared to nondiabetic mice. Administration of edaravone significantly attenuated diabetes induced RGCs death, upregulation of ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF. These findings suggest that oxidative stress plays a pivotal role in diabetic retinal damage and that systemic administration of edaravone may slow the progression of retinal neuropathy induced by diabetes.

  8. Prognostic significance of Fas and Fas ligand system-associated apoptosis in gastric cancer.

    Science.gov (United States)

    Ohno, S; Tachibana, M; Shibakita, M; Dhar, D K; Yoshimura, H; Kinugasa, S; Kubota, H; Masunaga, R; Nagasue, N

    2000-12-01

    Previous studies indicate that gastric carcinomas express Fas ligand and down-regulate Fas to escape from the host immune attack; however, the prognostic importance of Fas/FasL expression in this tumor is yet to be evaluated. Specimens from 87 gastric carcinoma patients of different stages treated in a defined period with curative intent were evaluated for apoptosis, Fas, FasL, and CD8 expression using an immunohistochemical method. The percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic cells expressed as apoptotic index (AI) was higher in 43 patients when the cut-off value was set at the median value. There were no significant correlations between AI and clinicopathologic parameters. Thirty-nine patients showed a high number of CD8+ cells within cancer nests. Positive FasL and Fas expression was seen in 53 and 72 patients, respectively. CD8 and FasL expressions were related only to patients' age. Fas expression had significant correlations with tumor invasion and Lauren classification. There were significant direct correlations between AI and number of nest CD8+ cells and between AI and grade of Fas expression. Apoptotic index, pT stage, CD8 expression, and Fas expression were identified as independent prognostic factors. Spontaneous apoptosis in gastric carcinoma may be an independent prognosticator for survival and is significantly influenced by tumor Fas expression and number of nest CD8 + cells.

  9. Ethanol extract of Portulaca Oleracea L. reduced the carbon tetrachloride induced liver injury in mice involving enhancement of NF-κB activity

    Science.gov (United States)

    Shi, Hongguang; Liu, Xuefeng; Tang, Gusheng; Liu, Haiyan; Zhang, Yinghui; Zhang, Bo; Zhao, Xuezhi; Wang, Wanyin

    2014-01-01

    Acute hepatic injury causes high morbidity and mortality world-wide. Management of severe acute hepatic failure continues to be one of the most challenging problems in clinical medicine. In present study, carbon tetrachloride (CCl4) was used to induce acute liver damage in mice and the protective effects of ethanol extract of Portulaca Oleracea L. (PO) were examined. The aminotransferase activities were biochemical estimated and the liver damage was tested by morphological histological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The role of PO on the activity of NF-κB was determined by luciferase reporter gene assay and immunohistochemistry. The level of p-p65 was tested by western blot. Our results showed that PO administration on mice would decrease the serum aminotransferase level and reduced the liver histological damage. We also found that nuclear translocation of p65 was enhanced in liver tissues of mice treated with PO compared with control animals. In addition, in cultured hepatic cells, PO increased the NF-κB luciferase reporter gene activity and upregulated the level of phosphorylation of p65, but had no effects on mice liver SOD activity and MDA level. Collectively, PO attenuated CCl4 induced mice liver damage by enhancement of NF-κB activity. PMID:25628785

  10. Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ.

    Science.gov (United States)

    Shin, Eun-Joo; Duong, Chu Xuan; Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F; Kanthasamy, Anumantha G; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2012-06-15

    This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (-/-) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (-/-) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (-/-) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Current research on methamphetamine-induced neurotoxicity: animal models of monoamine disruption.

    Science.gov (United States)

    Kita, Taizo; Wagner, George C; Nakashima, Toshikatsu

    2003-07-01

    Methamphetamine (METH)-induced neurotoxicity is characterized by a long-lasting depletion of striatal dopamine (DA) and serotonin as well as damage to striatal dopaminergic and serotonergic nerve terminals. Several hypotheses regarding the mechanism underlying METH-induced neurotoxicity have been proposed. In particular, it is thought that endogenous DA in the striatum may play an important role in mediating METH-induced neuronal damage. This hypothesis is based on the observation of free radical formation and oxidative stress produced by auto-oxidation of DA consequent to its displacement from synaptic vesicles to cytoplasm. In addition, METH-induced neurotoxicity may be linked to the glutamate and nitric oxide systems within the striatum. Moreover, using knockout mice lacking the DA transporter, the vesicular monoamine transporter 2, c-fos, or nitric oxide synthetase, it was determined that these factors may be connected in some way to METH-induced neurotoxicity. Finally a role for apoptosis in METH-induced neurotoxicity has also been established including evidence of protection of bcl-2, expression of p53 protein, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), activity of caspase-3. The neuronal damage induced by METH may reflect neurological disorders such as autism and Parkinson's disease.

  12. Melatonin attenuates oxidative stress, liver damage and hepatocyte apoptosis after bile-duct ligation in rats.

    Science.gov (United States)

    Aktas, Cevat; Kanter, Mehmet; Erboga, Mustafa; Mete, Rafet; Oran, Mustafa

    2014-10-01

    The goal of this study was to evaluate the possible protective effects of melatonin against cholestatic oxidative stress, liver damage and hepatocyte apoptosis in the common rats with bile duct ligation (BDL). A total of 24 male Wistar albino rats were divided into three groups: control, BDL and BDL + received melatonin; each group contains eight animals. Melatonin-treated BDL rats received daily melatonin 100 mg/kg/day via intraperitoneal injection. The application of BDL clearly increased the malondialdehyde (MDA) levels and decreased the superoxide dismutase (SOD) and glutathione (GSH) activities. Melatonin treatment significantly decreased the elevated tissue MDA levels and increased the reduced SOD and GSH enzyme levels in the tissues. The changes demonstrate that the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells and neutrophil infiltration into the widened portal areas as observed in the BDL group. The data indicate that melatonin attenuates BDL-induced cholestatic liver injury, bile duct proliferation and fibrosis. The α-smooth muscle actin (α-SMA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the BDL were observed to be reduced with the melatonin treatment. These results suggest that administration of melatonin is a potentially beneficial agent to reduce liver damage in BDL by decreasing oxidative stress. © The Author(s) 2012.

  13. 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.

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

    International Nuclear Information System (INIS)

    Shi, Yu; Zhao, Shuhua; Li, Jiejing; Mao, Bingyu

    2009-01-01

    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.

  15. Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary.

    Science.gov (United States)

    Morais, Roberto D V S; Thomé, Ralph G; Santos, Hélio B; Bazzoli, Nilo; Rizzo, Elizete

    2016-04-01

    In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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    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

  17. Protective Effects of Soy Oligopeptides in Ultraviolet B-Induced Acute Photodamage of Human Skin

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    Bing-rong Zhou

    2016-01-01

    Full Text Available Aim. We explored the effects of soy oligopeptides (SOP in ultraviolet B- (UVB- induced acute photodamage of human skin in vivo and foreskin ex vivo. Methods. We irradiated the forearm with 1.5 minimal erythemal dose (MED of UVB for 3 consecutive days, establishing acute photodamage of skin, and topically applied SOP. Erythema index (EI, melanin index, stratum corneum hydration, and transepidermal water loss were measured by using Multiprobe Adapter 9 device. We irradiated foreskin ex vivo with the same dose of UVB (180 mJ/cm2 for 3 consecutive days and topically applied SOP. Sunburn cells were detected by using hematoxylin and eosin staining. Apoptotic cells were detected by using terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Cyclobutane pyrimidine dimers (CPDs, p53 protein, Bax protein, and Bcl-2 protein were detected by using immunohistochemical staining. Results. Compared with UVB group, UVB-irradiated skin with topically applied SOP showed significantly decreased EI. Compared with UVB group, topical SOP significantly increased Bcl-2 protein expression and decreased CPDs-positive cells, sunburn cells, apoptotic cells, p53 protein expression, and Bax protein expressions in the epidermis of UVB-irradiated foreskin. Conclusion. Our study demonstrated that topical SOP can protect human skin against UVB-induced photodamage.

  18. Curcumin Protects against UVB-Induced Skin Cancers in SKH-1 Hairless Mouse: Analysis of Early Molecular Markers in Carcinogenesis

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    Kuen-Daw Tsai

    2012-01-01

    Full Text Available Curcumin (CUR has been shown to possess a preventive effect against various cancers and interfere with multiple-cell signaling pathways. We evaluated the protective effects of CUR in regression of UVB-induced skin tumor formation in SKH-1 hairless mice and its underlying early molecular biomarkers associated with carcinogenesis. Mice irradiated with UVB at 180 mJ/cm2 twice per week elicited 100% tumor incidence at 20 weeks. Topical application of CUR prior to UVB irradiation caused delay in tumor appearance, multiplicity, and size. Topical application of CUR prior to and immediately after a single UVB irradiation (180 mJ/cm2 resulted in a significant decrease in UVB-induced thymine dimer-positive cells, expression of proliferative cell nuclear antigen (PCNA, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase in p53 and p21/Cip1-positive cell population in epidermis. Simultaneously, CUR also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2, prostaglandin E2 (PGE2, and nitric oxide (NO levels. The results suggest that the protective effect of CUR against photocarcinogenesis is accompanied by downregulation of cell proliferative controls, involving thymine dimer, PCNA, apoptosis, transcription factors NF-κB, and of inflammatory responses involving COX-2, PGE2, and NO, while upregulation of p53 and p21/Cip1 to prevent DNA damage and facilitate DNA repair.

  19. Chlorella vulgaris triggers apoptosis in hepatocarcinogenesis-induced rats*

    Science.gov (United States)

    Mohd Azamai, Emey Suhana; Sulaiman, Suhaniza; Mohd Habib, Shafina Hanim; Looi, Mee Lee; Das, Srijit; Abdul Hamid, Nor Aini; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

    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. PMID:19198018

  20. Population control of resident and immigrant microglia by mitosis and apoptosis.

    Science.gov (United States)

    Wirenfeldt, Martin; Dissing-Olesen, Lasse; Anne Babcock, Alicia; Nielsen, Marianne; Meldgaard, Michael; Zimmer, Jens; Azcoitia, Iñigo; Leslie, Robert Graham Quinton; Dagnaes-Hansen, Frederik; Finsen, Bente

    2007-08-01

    Microglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have investigated the mechanisms of microglial population control in a well-defined model of reactive microgliosis in the mouse dentate gyrus after perforant pathway axonal lesion. Unbiased stereological methods and flow cytometry demonstrate significant lesion-induced increases in microglial numbers. Reactive microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations.

  1. Dysregulated IER3 Expression is Associated with Enhanced Apoptosis in Titin-Based Dilated Cardiomyopathy

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    Qifeng Zhou

    2017-03-01

    Full Text Available Apoptosis (type I programmed cell death of cardiomyocytes is a major process that plays a role in the progression of heart failure. The early response gene IER3 regulates apoptosis in a wide variety of cells and organs. However, its role in heart failure is largely unknown. Here, we investigate the role of IER3 in an inducible heart failure mouse model. Heart failure was induced in a mouse model that imitates a human titin truncation mutation we found in a patient with dilated cardiomyopathy (DCM. Transferase dUTP nick end labeling (TUNEL and ssDNA stainings showed induction of apoptosis in titin-deficient cardiomyocytes during heart failure development, while IER3 response was dysregulated. Chromatin immunoprecipitation and knock-down experiments revealed that IER3 proteins target the promotors of anti-apoptotic genes and act as an anti-apoptotic factor in cardiomyocytes. Its expression is blunted during heart failure development in a titin-deficient mouse model. Targeting the IER3 pathway to reduce cardiac apoptosis might be an effective therapeutic strategy to combat heart failure.

  2. Application of Photoshop-based image analysis and TUNEL for the distribution and quantification of dexamethasone-induced apoptotic cells in rat thymus.

    Science.gov (United States)

    Hussar, Piret; Tokin, Ivan; Hussar, Ulo; Filimonova, Galina; Suuroja, Toivo

    2006-01-01

    The aim of the present study was to determine the target site cells in the rat thymus after exposure to the synthetic glucocorticoid, dexamethasone, at therapeutic doses. The findings of histology and histochemistry (Feulgen, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling--TUNEL) with quantification by computerized histomorphometry are described. A quantified investigation of apoptotic and mitotic thymic lymphocytes in 36 young adult Wistar rats was performed at 1-7 days after a 3-day injection of dexamethasone (a total dose of 1.2 mg/rat intraperitoneally). At the first day after dexamethasone administration the moderate involution and atrophy of thymus histology were observed with simultaneous fall in cortical cellularity and mitotic activity of thymocytes. More rapid fall appeared in the inner cortex. The number of apoptotic (TUNEL-positive) cells was significantly increased. On the days 5 and 7 the expression of apoptosis and the cell proliferation were at almost normal level. The findings suggest that dexamethasone-induced apoptosis of cortical thymic lymphocytes, mainly correlated with synchronous inhibition of mitosis and cell number fall in thymus. The main target sites of dexamethasone injury were cells in the inner cortex of lobuli thymi.

  3. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Vitrification of neat semen alters sperm parameters and DNA integrity.

    Science.gov (United States)

    Khalili, Mohammad Ali; Adib, Maryam; Halvaei, Iman; Nabi, Ali

    2014-05-06

    Our aim was to evaluate the effect of neat semen vitrification on human sperm vital parameters and DNA integrity in men with normal and abnormal sperm parameters. Semen samples were 17 normozoospermic samples and 17 specimens with abnormal sperm parameters. Semen analysis was performed according to World Health Organization (WHO) criteria. Then, the smear was provided from each sample and fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Vitrification of neat semen was done by plunging cryoloops directly into liquid nitrogen and preserved for 7 days. The samples were warmed and re-evaluated for sperm parameters as well as DNA integrity. Besides, the correlation between sperm parameters and DNA fragmentation was assessed pre- and post vitrification. Cryopreserved spermatozoa showed significant decrease in sperm motility, viability and normal morphology after thawing in both normal and abnormal semen. Also, the rate of sperm DNA fragmentation was significantly higher after vitrification compared to fresh samples in normal (24.76 ± 5.03 and 16.41 ± 4.53, P = .002) and abnormal (34.29 ± 10.02 and 23.5 ± 8.31, P < .0001), respectively. There was negative correlation between sperm motility and sperm DNA integrity in both groups after vitrification. Vitrification of neat ejaculates has negative impact on sperm parameters as well as DNA integrity, particularly among abnormal semen subjects. It is, therefore, recommend to process semen samples and vitrify the sperm pellets.

  6. Cellular changes in the hamster testicular interstitium with ageing and after exposure to short photoperiod.

    Science.gov (United States)

    Beltrán-Frutos, E; Seco-Rovira, V; Ferrer, C; Madrid, J F; Sáez, F J; Canteras, M; Pastor, L M

    2016-04-01

    The aim of this study was to evaluate the cellular changes that occur in the hamster testicular interstitium in two very different physiological situations involving testicular involution: ageing and exposure to a short photoperiod. The animals were divided into an 'age group' with three subgroups - young, adult and old animals - and a 'regressed group' with animals subjected to a short photoperiod. The testicular interstitium was characterised by light and electron microscopy. Interstitial cells were studied histochemically with regard to their proliferation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling (TUNEL+) and testosterone synthetic activity. We identified two types of Leydig cell: Type A cells showed a normal morphology, while Type B cells appeared necrotic. With ageing, pericyte proliferation decreased but there was no variation in the index of TUNEL-positive Leydig cells. In the regressed group, pericyte proliferation was greater and TUNEL-positive cells were not observed in the interstitium. The testicular interstitium suffered few ultrastructural changes during ageing and necrotic Leydig cells were observed. In contrast, an ultrastructural involution of Leydig cells with no necrosis was observed in the regressed group. In conclusion, the testicular interstitium of Mesocricetus auratus showed different cellular changes in the two groups (age and regressed), probably due to the irreversible nature of ageing and the reversible character of changes induced by short photoperiod.

  7. Correlation between Gene Expression and Osteoarthritis Progression in Human.

    Science.gov (United States)

    Zhong, Leilei; Huang, Xiaobin; Karperien, Marcel; Post, Janine N

    2016-07-14

    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.

  8. 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.

  9. [Effect of jiaotai pill on pancreatic fat accumulation and islet cell apoptosis in rats with type 2 diabetes].

    Science.gov (United States)

    Zou, Xin; Liu, De-Liang; Lu, Fu-Er; Dong, Hui; Xu, Li-Jun; Luo, Yun-Huan; Wang, Kai-Fu

    2014-06-01

    In this study, the rat type 2 diabetes mellitus (T2DM) model was established through tail vein injection with low dose of streptozotocin (STZ) and high fat diet for 8 weeks, and then treated with Jiaotai Pill. The oral glucose tolerance test (OGTT), fasting serum insulin (FINS), free fatty acid(FFA) levels and blood lipid were assayed. HOMA-IR was calculated. Pancreatic pathology was performed. And pancreatic triglyceride (TG) content was examined by the lipid extraction method. Pancreatic islet cell apoptosis were detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). According to the results, the model group showed abnormal OGTT, increased FINS, HOMA-IR, FFA, lipid disorder, obvious fat accumulation and significantly increased TG content in pancreatic tissues, and enhanced pancreatic islet cell apoptosis. Compared with the model group, the Jiaotai Pill group displayed improved OGTT, reduced FINS, HOMA-IR, FFA, recovered lipid disorder, decreased fat accumulation and significantly declined TG content in pancreatic tissues, and lowered pancreatic islet cell apoptosis. In summary, Jiaotai pill could effectively treat type 2 diabetes in rats. Its mechanism may be related to the reduction in pancreatic fat accumulation and islet cell apoptosis.

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

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    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.

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

    Science.gov (United States)

    Hong, S.; Hara, H.; Shimazawa, M.; Hyakkoku, K.; Kim, C.Y.; Seong, G.J.

    2012-01-01

    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 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. PMID:22331138

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

    International Nuclear Information System (INIS)

    Kim, Mi Sun; Lee, Eun Jung; Kim, Jae Won; Keum, Ki Chang; Koom, Woong Sub; Chung, Ui Seok; Koh, Won Gun

    2016-01-01

    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

  13. Human Scalp Hair Follicles Are Both a Target and a Source of Prolactin, which Serves as an Autocrine and/or Paracrine Promoter of Apoptosis-Driven Hair Follicle Regression

    Science.gov (United States)

    Foitzik, Kerstin; Krause, Karoline; Conrad, Franziska; Nakamura, Motonobu; Funk, Wolfang; Paus, Ralf

    2006-01-01

    The prototypic pituitary hormone prolactin (PRL) exerts a wide variety of bioregulatory effects in mammals and is also found in extrapituitary sites, including murine skin. Here, we show by reverse transcriptase-polymerase chain reaction and immunohistology that, contrary to a previous report, human skin and normal human scalp hair follicles (HFs), in particular, express both PRL and PRL receptors (PRL-R) at the mRNA and protein level. PRL and PRL-R immunoreactivity can be detected in the epithelium of human anagen VI HFs, while the HF mesenchyme is negative. During the HF transformation from growth (anagen) to apoptosis-driven regression (catagen), PRL and PRL-R immunoreactivity appear up-regulated. Treatment of organ-cultured human scalp HFs with high-dose PRL (400 ng/ml) results in a significant inhibition of hair shaft elongation and premature catagen development, along with reduced proliferation and increased apoptosis of hair bulb keratinocytes (Ki-67/terminal dUTP nick-end labeling immunohistomorphometry). This shows that PRL receptors, expressed in HFs, are functional and that human skin and human scalp HFs are both direct targets and sources of PRL. Our data suggest that PRL acts as an autocrine hair growth modulator with catagen-promoting functions and that the hair growth-inhibitory effects of PRL demonstrated here may underlie the as yet ill-understood hair loss in patients with hyperprolactinemia. PMID:16507890

  14. Neurotoxic, cytotoxic, apoptotic and antiproliferative effects of some marine algae extracts on the NA2B cell line.

    Science.gov (United States)

    Kurt, O; Özdal-Kurt, F; Akçora, C M; Özkut, M; Tuğlu, M I

    2018-02-01

    Oxidative stress contributes to cancer pathologies and to apoptosis. Marine algae exhibit cytotoxic, antiproliferative and apoptotic effects; their metabolites have been used to treat many types of cancer. We investigated in culture extracts of Petalonia fascia, Jania longifurca and Halimeda tuna to determine their effects on mouse neuroblastoma cell line, NA2B. NA2B cells were treated with algae extracts, and the survival and proliferation of NA2B cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of algae extracts on oxidative stress in NA2B cells also were investigated using nitric oxide synthase (NOS) immunocytochemistry and apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling. We observed significant neurite inhibition with moderate damage by the neurotoxicity-screening test (NST) at IC 50 dilutions of the extracts. MTT demonstrated that J. longifurca extracts were more toxic than P. fascia and H. tuna extracts. We found an increase of endothelial and inducible NOS immunostaining for oxidative stress and TUNEL analysis revealed increased apoptosis after application of extract. Our findings suggest that the algae we tested may have potential use for treatment of cancer.

  15. Effects of Hormone Deprivation, 2-Methoxyestradiol Combination Therapy on Hormone-Dependent Prostate Cancer In Vivo

    Directory of Open Access Journals (Sweden)

    Fuminori Sato

    2005-09-01

    Full Text Available 2-Methoxyestradiol (2-ME has potent anti proliferative effects on cancer cells. Its utility alone or in combination with other therapies for treating prostate cancer, however, has not been fully explored. Androgendependent, independent human prostate cancer cells were examined in vivo for their response to combination therapy. Efficacy was assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, measuring microvessel density (MVD in excised tumors. Animals harboring hormonedependent tumors treated with 2-ME alone, androgen deprivation therapy alone, or the combination of the two had a 3.1-fold, 5.3-fold, 10.1-fold increase in apoptosis, respectively. For hormone-independent tumors, treatment with 2-ME resulted in a 2.43-fold increase in apoptosis, a 73% decrease in MVD. 2-ME was most effective against hormone-dependent tumors in vivo, combination therapy resulted in a significant increase in efficacy compared to no treatment controls, trended toward greater efficacy than either 2-ME or androgen deprivation alone. Combination therapy should be investigated further as an additional therapeutic option for early prostate cancer.

  16. Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

    International Nuclear Information System (INIS)

    Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

    2015-01-01

    To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis

  17. Heme oxygenase-1 prevents hyperthyroidism induced hepatic damage via an antioxidant and antiapoptotic pathway.

    Science.gov (United States)

    Giriş, Murat; Erbil, Yeşim; Depboylu, Bilge; Mete, Ozgür; Türkoğlu, Umit; Abbasoğlu, Semra Doğru; Uysal, Müjdat

    2010-12-01

    The exact pathogenesis of hepatic dysfunction in hyperthyroidism is still unknown. We aimed to investigate the pathogenesis of liver dysfunction caused by hyperthyroidism through inducing heme oxygenase-1 (HO-1) expression, which has antioxidant and anti-apoptotic properties. Rats were divided into six groups: untreated (group 1), treated with zinc protoporphyrin (ZnPP) (group 2), treated with hemin (group 3), treated with tri-iodothyronine (T3) (group 4), treated with T3 and ZnPP (group 5), and treated with T3 and hemin (group 6). After 22 d, oxidative stress and antioxidant enzymes and the expression of HO-1, mitochondrial permeability transition, cytochrome c, Bax, Bcl-2, caspase-3, caspase-8, and caspase-3 activity, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were examined. Hyperthyroidism induced oxidative stress of liver tissue was ameliorated by HO-1 induction. Administration of hemin (HO-1 inducer) increased Bcl-2 expression. Decreased expression of cytochrome c was accompanied by a decrease in caspase-3, caspase-8, Bax expression, and caspase-3 activity. The apoptotic activity and oxidative damage were found to be increased by the administration of ZnPP (HO-1 inhibitor). Immunohistochemistry findings supported these results. HO-1 induction plays a protective role in the pathogenesis of the liver dysfunction in hyperthyroidism. This effect is dependent on modulation of the antiapoptotic and antioxidative pathways by HO-1 expression. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Opposite effects of the gap junction blocker octanol on focal cerebral ischemia occluded for different durations.

    Science.gov (United States)

    Ding, Wenting; Zhou, Lequan; Liu, Wei; Guan, Li; Li, Xiaoying; Liu, Haimei; Yan, Fuman; Xu, Jinwen; Zeng, Weiyong; Qiu, Min

    2014-06-01

    Protectants and executioners have been demonstrated to be used by gap junctions in focal cerebral ischemia. Certain researchers hypothesized that the opposite role of gap junctions may be associated with the injury extent, which has been demonstrated to be highly correlated with occlusion duration. In order to examine this hypothesis directly, the effects of octanol, a frequently used drug, were examined to investigate the role of gap junctions, in rats following middle cerebral artery occlusion (MCAO) for 30 min/2 h and 24 h reperfusion, respectively. Octanol significantly reduced the infarct volume following 2 h of occlusion concomitant with lower neurological deficits, whereas it enlarged the infarct volume following 30 min of occlusion. Consistently, octanol attenuated the number of transferase dUTP nick-end labeling (TUNEL) positive neurons in the hippocampal CA1 region following 2 h of occlusion, while opposite effects were observed for 30 min of occlusion. Further immunohistochemical studies demonstrated that the expression of B-cell leukemia-2 (Bcl-2, anti-apoptotic protein) was upregulated and that Bcl-2-associated X (Bax, proapoptotic protein) was downregulated following 2 h of occlusion in the octanol group compared with the ischemic group. Conversely, octanol downregulated the expression of the Bcl-2 protein concomitant with increased Bax protein following 30 min of occlusion. These results indicated that the gap junction blocker octanol can protect against ischemic injury following long-term occlusion, however, can aggravate ischemic injury following short-term occlusion.

  19. The Anticancer Effects of Radachlorin-mediated Photodynamic Therapy in the Human Endometrial Adenocarcinoma Cell Line HEC-1-A.

    Science.gov (United States)

    Kim, Su-Mi; Rhee, Yun-Hee; Kim, Jong-Soo

    2017-11-01

    We investigated the effect of photodynamic therapy (PDT) using radachlorin on invasion, vascular formation and apoptosis by targeting epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in the HEC-1-A endometrial adenocarcinoma cell line. To investigate the apoptotic pathway, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and western blot analysis. We also evaluated the effects of PDT on tubular capillary formation in and invasion by HEC-1-A cells with a tube formation assay, invasion assay, prostaglandin E2 (PGE2) assay, and western blot analysis. PDT had anticancer effects on HEC-1-A through activation of the intrinsic pathway of apoptosis via caspase-9 and poly-(ADP-ribose) polymerase (PARP). PDT also inhibited tubular capillary formation in and invasion by HEC-1-A under VEGF pretreatment, that resulted from down-regulation of VEGFR2, EGFR, Ras homolog gene family/ member A (RhoA) and PGE2. These results are indicative of the specificity of radachlorin-mediated PDT to VEGF. The major advantage of radachlorin-mediated PDT is its selectivity for cancer tissue while maintaining adjacent normal endometrial tissue. Therefore, radachlorin-mediated PDT might offer high anticancer efficacy for endometrial adenocarcinoma and an especially useful modality for preserving fertility. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  20. Prenatal ionizing radiation-induced apoptosis of the developing murine brain with special references to the expression of some proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Makiko; Itoh, Kyoko; Matsumoto, Akira; Hayashi, Yoshitake; Sasaki, Ryohei; Imai, Yukihiro; Itoh, Hiroshi [Kobe Univ. (Japan). School of Medicine

    2001-04-01

    Apoptosis induced by ionizing irradiation of the developing mouse brain was investigated by using histology, analysis of DNA fragmentation on agarose gel and electron microscopy. A TUNEL-labeled index (L.I.) was calculated from the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay in 4 specific regions, cortical plate, intermediate zone, subependymal zone, and subependymal germinal matrix. The kinetics of apoptosis associated protein was examined by western blotting and immunofluorescence. C57BL/6J mice pregnant on embryonic day 14 (E14) were exposed to a single dose of 1.5-Gy irradiation. Irradiaited fetal brains at E15 and E17 showed extensive apoptosis with morphological characteristics. In all 4 regions, L.I. was greater in irradiated brains than in control brains at E15 and E17. Most of TUNEL-labeled cells expressed a mature neuronal marker (NeuN) and Bax protein, which is up-regulated in irradiation-induced apoptosis. Ionizing radiation moderately enhanced expression of Bax, Bcl-xL, and Cpp32 proteins. Postnatal irradiated mice showed microencephaly as compared to age-matched mice and the weight of whole body including brain decreased moderately. (author)

  1. Prenatal ionizing radiation-induced apoptosis of the developing murine brain with special references to the expression of some proteins

    International Nuclear Information System (INIS)

    Kitamura, Makiko; Itoh, Kyoko; Matsumoto, Akira; Hayashi, Yoshitake; Sasaki, Ryohei; Imai, Yukihiro; Itoh, Hiroshi

    2001-01-01

    Apoptosis induced by ionizing irradiation of the developing mouse brain was investigated by using histology, analysis of DNA fragmentation on agarose gel and electron microscopy. A TUNEL-labeled index (L.I.) was calculated from the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay in 4 specific regions, cortical plate, intermediate zone, subependymal zone, and subependymal germinal matrix. The kinetics of apoptosis associated protein was examined by western blotting and immunofluorescence. C57BL/6J mice pregnant on embryonic day 14 (E14) were exposed to a single dose of 1.5-Gy irradiation. Irradiated fetal brains at E15 and E17 showed extensive apoptosis with morphological characteristics. In all 4 regions, L.I. was greater in irradiated brains than in control brains at E15 and E17. Most of TUNEL-labeled cells expressed a mature neuronal marker (NeuN) and Bax protein, which is up-regulated in irradiation-induced apoptosis. Ionizing radiation moderately enhanced expression of Bax, Bcl-xL, and Cpp32 proteins. Postnatal irradiated mice showed microencephaly as compared to age-matched mice and the weight of whole body including brain decreased moderately. (author)

  2. Studying effects of Magnolol on alpha-particle induced bystander effects using PADC-film based dishes

    International Nuclear Information System (INIS)

    Wong, T.P.W.; Tse, A.K.W.; Fong, W.F.; Yu, K.N.

    2009-01-01

    Radiation-induced bystander effect refers to the biological response found in cells (called bystander cells) which are not irradiated directly by ionizing radiation but are next to cells irradiated directly by ionizing radiation. In the present paper, the effects of Magnolol, an extract from the bark of Magnolia officinalis which is used as a traditional Chinese medicine, were studied on alpha-particle induced bystander effects. In our experiments, Chinese hamster ovary (CHO) cells were cultured in PADC-film based dishes and were irradiated with low fluences of alpha particles passing through the PADC films. The precise number of cells traversed or missed by alpha particles could be determined by studying the alpha-particle tracks developed on the PADC films upon subsequent chemical etching. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was employed to analyze the biological response of bystander cells in terms of DNA strand breaks. With the pretreatment of Magnolol, the DNA strand breaks in bystander cells were reduced, which showed that the alpha-particle induced bystander effects were suppressed with the presence of Magnolol. Since Magnolol is an antioxidant which can scavenge reactive oxygen species (ROS), our results give support to that ROS play a role in the bystander signal transmission in our experiments.

  3. Fisetin Protects against Intracerebral Hemorrhage-Induced Neuroinflammation in Aged Mice.

    Science.gov (United States)

    Chen, Cheng; Yao, Li; Cui, Jing; Liu, Bao

    2018-01-01

    Fisetin is commonly used as an anti-inflammatory and neuroprotective drug. In this study, we aimed to investigate the efficacy of fisetin in alleviating intracerebral hemorrhage (ICH)-induced brain injury. Mouse ICH models were constructed using the collagenase-induction method. ICH mice received fisetin treatment at the dose of 10-90 mg/kg, followed by the evaluation of neurological deficit through neurologic severity scores (mNSS), brain water content and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis of cell apoptosis. Cytokine levels were also assessed with enzyme-linked immunosorbent assay. The activation of astrocytes and microglia was evaluated through S100 staining and Western blot analysis of ionized calcium-binding adaptor molecule 1 respectively. Nuclear factor kappa-B (NF-κB) signaling was also evaluated by Western blot. ICH mice demonstrated dramatic increase in mNSS, brain edema and cell apoptosis, indicating severe brain deficit. Fisetin treatment lowered these parameters, suggesting the alleviation of brain injury. Levels of proinflammatory cytokines were reduced, accompanied by a prominent decrease in activated astrocytes and microglia. NF-κB signaling was also attenuated by fisetin treatment. Fisetin effectively alleviates ICH by downregulating proinflammatory cytokines and attenuating NF-κB signaling. These data suggest fisetin as a valuable natural flavonol for clinical management of ICH-induced brain injury. © 2018 S. Karger AG, Basel.

  4. Suppression of heat shock protein 70 by siRNA enhances the antitumor effects of cisplatin in cultured human osteosarcoma cells.

    Science.gov (United States)

    Mori, Yuki; Terauchi, Ryu; Shirai, Toshiharu; Tsuchida, Shinji; Mizoshiri, Naoki; Arai, Yuji; Kishida, Tsunao; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

    2017-09-01

    Although advances in chemotherapy have improved the prognosis for osteosarcoma, some patients do not respond sufficiently to treatment. Heat shock protein 70 (Hsp70) is expressed at high levels in cancer cells and attenuates the therapeutic efficacy of anticancer agents, resulting in a poorer prognosis. This study investigated whether small interfering RNA (siRNA)-mediated inhibition of Hsp70 expression in an osteosarcoma cell line would enhance sensitivity to cisplatin. The expression of Hsp70 with cisplatin treatment was observed by using Western blotting and real-time reverse transcription polymerase chain reaction (RT-PCR). Changes in the IC 50 of cisplatin when Hsp70 was inhibited by siRNA were evaluated. Cisplatin's effectiveness in inducing apoptosis was assessed by assay of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), caspase-3 activity, and mitochondrial membrane potential. Up-regulation of Hsp70 expression was dependent on the concentration of cisplatin. Inhibition of Hsp70 expression significantly reduced the IC 50 of cisplatin. When cisplatin was added to osteosarcoma cells with Hsp70 expression inhibited, a significant increase in apoptosis was demonstrated in TUNEL, caspase-3, and mitochondrial membrane potential assays. Inhibition of Hsp70 expression induced apoptosis in cultured osteosarcoma cells, indicating that Hsp70 inhibition enhanced sensitivity to cisplatin. Inhibition of Hsp70 expression may provide a new adjuvant therapy for osteosarcoma.

  5. Melatonin mitigates neomycin-induced hair cell injury in zebrafish.

    Science.gov (United States)

    Oh, Kyoung Ho; Rah, Yoon Chan; Hwang, Kyu Ho; Lee, Seung Hoon; Kwon, Soon Young; Cha, Jae Hyung; Choi, June

    2017-10-01

    Ototoxicity due to medications, such as aminoglycosides, is irreversible, and free radicals in the inner ear are assumed to play a major role. Because melatonin has an antioxidant property, we hypothesize that it might mitigate hair cell injury by aminoglycosides. The objective of this study was to evaluate whether melatonin has an alleviative effect on neomycin-induced hair cell injury in zebrafish (Danio rerio). Various concentrations of melatonin were administered to 5-day post-fertilization zebrafish treated with 125 μM neomycin for 1 h. Surviving hair cells within four neuromasts were compared with that of a control group. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The changes of ultrastructure were confirmed using a scanning electron microscope. Melatonin alleviated neomycin-induced hair cell injury in neuromasts (neomycin + melatonin 100 μM: 13.88 ± 0.91 cells, neomycin only: 7.85 ± 0.90 cells; n = 10, p melatonin for 1 h in SEM findings. Melatonin is effective in alleviating aminoglycoside-induced hair cell injury in zebrafish. The results of this study demonstrated that melatonin has the potential to reduce apoptosis induced by aminoglycosides in zebrafish.

  6. 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.

  7. Protective effect of Urtica dioica on liver damage induced by biliary obstruction in rats.

    Science.gov (United States)

    Oguz, Serhat; Kanter, Mehmet; Erboga, Mustafa; Ibis, Cem

    2013-10-01

    The aim of this study was to evaluate the possible protective effects of Urtica dioica (UD) against liver damage in the common bile duct-ligated rats. A total of 24 male Sprague Dawley rats were divided into three groups, namely, control, bile duct ligation (BDL) and BDL + received UD groups, containing eight animals in each group. The rats in UD-treated groups were given UD oils (2 ml/kg) once a day intraperitoneally for 2 weeks starting 3 days prior to BDL operation. The change demonstrating the bile duct proliferation and fibrosis in expanded portal tracts includes the extension of proliferated bile ducts into the lobules; inflammatory cell infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with UD attenuated alterations in liver histology. The α-smooth muscle actin, cytokeratin-positive ductular proliferation and the activity of terminal deoxynucleotidyl transferase dUTP nick end labeling in the BDL were observed to be reduced with the UD treatment. The data indicate that UD attenuates BDL-induced cholestatic liver injury, bile duct proliferation and fibrosis.

  8. Protective effects of Urtica dioica L. on experimental testicular ischaemia reperfusion injury in rats.

    Science.gov (United States)

    Aktas, C; Erboga, M; Fidanol Erboga, Z; Bozdemir Donmez, Y; Topcu, B; Gurel, A

    2017-05-01

    In this study, it was aimed to examine the effects of Urtica dioica L. (UD) that has antioxidant feature in the experimental testicular I/R model in rats in terms of anti-apoptotic and antioxidative effects. In our study, 24 male rats were divided into three groups: control group, I/R group and I/R + UD (2 mg kg -1 ) group. Seminiferous tubule calibre measurement, Johnson score, haematoxylin-eosin staining, proliferative cell nucleus antigen (PCNA) immunohistochemical staining and TUNEL as histopathological have been conducted. The structural deterioration in the testicular on I/R group has reduced after the treatment of UD. Our data indicate a significant reduction in the activity of in situ identification of apoptosis using terminal dUTP nick end labelling (TUNEL), and there was a rise in the expression of proliferating cell nuclear antigen (PCNA) in testis tissues of UD-treated rats in the I/R group. The I/R + UD group showed a decrease in malondialdehyde levels and an increase in the activities of superoxide dismutase, catalase and glutathione peroxidase in comparison with the I/R group. It could be concluded that protective effects of UD on the I/R testicles are via reduction of histological damage, apoptosis, oxidative stress and lipid peroxidation. © 2016 Blackwell Verlag GmbH.

  9. Effects of Urtica dioica dichloromethane extract on cell apoptosis and related gene expression in human breast cancer cell line (MDA-MB-468).

    Science.gov (United States)

    Mohammadi, A; Mansoori, B; Goldar, S; Shanehbandi, D; Khaze, V; Mohammadnejad, L; Baghbani, E; Baradaran, B

    2016-02-29

    Breast cancer is the most common cancer among women in worldwide, especially in developing countries. Therefore, a large number of anticancer agents with herbal origins have been reported against this deadly disease. This study is the first to examine the cytotoxic and apoptotic effects of Urtica dioica in MDA-MB-468, human breast adenocarcinoma cells. The 3-(4,5-dimethylethiazol-2 yl)-2,5- diphenyltetrazolium (MTT) reduction and trypan-blue exclusion assay were performed in MDA-MB-468 cells as well as control cell line L929 to analyze the cytotoxic activity of the dichloromethane extract. In addition, Apoptosis induction of Urtica dioica on the MDA-MB-468 cells was assessed using TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) assay and DNA fragmentation analysis and real-time polymerase chain reaction (PCR). The results showed that the extract significantly inhibited cell growth and viability without inducing damage to normal control cells. Nuclei Staining in TUNEL and DNA fragments in DNA fragmentation assay and increase in the mRNA expression levels of caspase-3, caspase-9, decrease in the bcl2 and no significant change in the caspase-8 mRNA expression level, showed that the induction of apoptosis was the main mechanism of cell death that induce by Urtica dioica extract. Our results suggest that urtica dioica dichloromethane extract may contain potential bioactive compound(s) for the treatment of breast adenocarcinoma.

  10. Studying effects of Magnolol on alpha-particle induced bystander effects using PADC-film based dishes

    Energy Technology Data Exchange (ETDEWEB)

    Wong, T.P.W. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Tse, A.K.W.; Fong, W.F. [Research and Development Division, School of Chinese Medicine, Hong Kong Baptist University, Baptist University Road, Kowloon Tong (Hong Kong); Yu, K.N., E-mail: peter.yu@cityu.edu.h [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong)

    2009-10-15

    Radiation-induced bystander effect refers to the biological response found in cells (called bystander cells) which are not irradiated directly by ionizing radiation but are next to cells irradiated directly by ionizing radiation. In the present paper, the effects of Magnolol, an extract from the bark of Magnolia officinalis which is used as a traditional Chinese medicine, were studied on alpha-particle induced bystander effects. In our experiments, Chinese hamster ovary (CHO) cells were cultured in PADC-film based dishes and were irradiated with low fluences of alpha particles passing through the PADC films. The precise number of cells traversed or missed by alpha particles could be determined by studying the alpha-particle tracks developed on the PADC films upon subsequent chemical etching. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was employed to analyze the biological response of bystander cells in terms of DNA strand breaks. With the pretreatment of Magnolol, the DNA strand breaks in bystander cells were reduced, which showed that the alpha-particle induced bystander effects were suppressed with the presence of Magnolol. Since Magnolol is an antioxidant which can scavenge reactive oxygen species (ROS), our results give support to that ROS play a role in the bystander signal transmission in our experiments.

  11. 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.

  12. Effect of low dose X-ray irradiation on apoptosis in spermatogenic cells of mouse testes

    International Nuclear Information System (INIS)

    Liu Guangwei; Liu Shuchun; Lu Zhe; Gong Shouliang

    2003-01-01

    To study the effects of low dose radiation (LDR) with different doses of X-rays on the apoptosis in spermatogenic cells of male Kunming mouse testes. The time-effect and dose-effect of apoptosis in the different stages of spermatogenic cell cycles of mouse testis after LDR with different doses of X-rays were studied with light microscope using the methods of TdT-mediated dUTP nick end labeling (TUNEL) and HE staining. The apoptosis of spermatogenic cells induced by LDR had a remarkable regularity in cell types. When the dose was 0.025 Gy, spermatogonium apoptosis was taken as main. With the dose increase of irradiation (0.025-0.2 Gy), spermatocytes also showed an apoptotic change, but the apoptotic rate of spermatogonia was significantly higher than that of spermatocytes. Moreover, the apoptosis of spermatids and spermatozoa scarcely occurred after irradiation with low dose. The apoptosis of spermatogenic cells induced by LDR has a regular change, which provides a further experimental evidence for the mechanism study of hormesis by LDR

  13. Depressed Immune Responses and Accelerated Splenic Apoptosis due to Experience of Food Deprivation and Inequality but not Unstable Social Status in Balb/c Mice.

    Science.gov (United States)

    Aghajani, Marjan; Vaez Mahdavi, Mohammad Reza; Najafabadi, Mohsen Khalili; Ghazanfari, Tooba; Moradi, Fatemeh; Golchoobian, Ravieh; Askari, Hasan; Sanadgol, Nima; Moghaddam, Ehsan Kazemi

    2017-01-01

    We aimed to show that the immune system is sensitive to the detrimental effects of inequality and social injustice, and splenic vulnerability to apoptosis may also increase. In order of better determination of immune responses to chronic social stress, we implemented food deprivation, food intake inequality, and unstable social status (a change of cage-mate every 3 days) for a period of 14 days in 60 male Balb/c mice. At the end of this stress period, nitric oxide (NO) production by peritoneal adherent cells and the serum concentration of corticosterone were measured. Moreover, the viability of peritoneal adherent cells and spleen lymphocytes was evaluated by MTT assay. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was done to reveal the TUNEL-reactive apoptotic bodies in the spleen. Our results showed that food deprivation and inequality caused significant changes in the apoptosis of splenic cells in comparison with the control group (p social status did not cause a further increase in the viability of lymphocytes and peritoneal adherent cells, or NO production in animals that were food-deprived or experienced inequality. Serum concentration of corticosterone in all experimental groups, except for animals that experienced unstable social status only, significantly decreased versus the control group (p poverty and social inequality, but not unstable social status, affect immune responses and are likely involved in the induction of splenic apoptosis in mice. © 2017 S. Karger AG, Basel.

  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. 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.

  16. Engraftment Site and Effectiveness of the Pan-Caspase Inhibitor F573 to Improve Engraftment in Mouse and Human Islet Transplantation in Mice.

    Science.gov (United States)

    Pepper, Andrew R; Bruni, Antonio; Pawlick, Rena; Wink, John; Rafiei, Yasmin; Gala-Lopez, Boris; Bral, Mariusz; Abualhassan, Nasser; Kin, Tatsuya; Shapiro, A M James

    2017-10-01

    Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice. Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing. F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P islet function when transplanted under the KC (P islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

  17. Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

    Science.gov (United States)

    Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

    2006-06-01

    This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

  18. Correlation of lung surface area to apoptosis and proliferation in human emphysema.

    Science.gov (United States)

    Imai, K; Mercer, B A; Schulman, L L; Sonett, J R; D'Armiento, J M

    2005-02-01

    Pulmonary emphysema is associated with alterations in matrix proteins and protease activity. These alterations may be linked to programmed cell death by apoptosis, potentially influencing lung architecture and lung function. To evaluate apoptosis in emphysema, lung tissue was analysed from 10 emphysema patients and six individuals without emphysema (normal). Morphological analysis revealed alveolar cells in emphysematous lungs with convoluted nuclei characteristic of apoptosis. DNA fragmentation was detected using terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) and gel electrophoresis. TUNEL revealed higher apoptosis in emphysematous than normal lungs. Markers of apoptosis, including active caspase-3, proteolytic fragment of poly (ADP-ribose) polymerase, Bax and Bad, were detected in emphysematous lungs. Linear regression showed that apoptosis was inversely correlated with surface area. Emphysematous lungs demonstrated lower surface areas and increased cell proliferation. There was no correlation between apoptosis and proliferation, suggesting that, although both events increase during emphysema, they are not in equilibrium, potentially contributing to reduced lung surface area. In summary, cell-based mechanisms associated with emphysematous parenchymal damage include increased apoptosis and cell proliferation. Apoptosis correlated with airspace enlargement, supporting epidemiological evidence of the progressive nature of emphysema. These data extend the understanding of cell dynamics and structural changes within the lung during emphysema pathogenesis.

  19. Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro.

    Science.gov (United States)

    Meller, R; Schindler, C K; Chu, X P; Xiong, Z G; Cameron, J A; Simon, R P; Henshall, D C

    2003-05-01

    Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

  20. Effect of GuiXiong Xiaoyi Wan in Treatment of Endometriosis on Rats

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    Zhixing Jin

    2015-01-01

    Full Text Available Objective. To evaluate the effect of GuiXiong Xiaoyi Wan (GXXYW on the development of endometriosis in a rat model. Methods. Sprague-Dawley rats with surgically induced endometriosis were randomly treated with low-dose GXXYW, high-dose GXXYW, or vehicle (negative control for 28 days. Immunohistochemistry was used to assess cell proliferation in the lesions. The terminal deoxynucleotidyl transferase- (TdT- mediated dUTP biotin nick end labelling (TUNEL method was performed to analyse the apoptosis induced by GuiXiong Xiaoyi Wan. The percentages of CD3+ lymphocytes, CD4+ lymphocytes, and CD8+ lymphocytes in the spleens of the rats were evaluated using flow cytometric analysis. Results. Treatment with GXXYW significantly decreased the lesion size, inhibited cell proliferation, and induced apoptosis in endometriotic tissue. The spleens of GXXYW-treated rats also demonstrated a significant increase in the percentage of CD4+ lymphocytes and a significant decrease in the percentage of CD8+ lymphocytes. Conclusions. These results suggest that, in a rat model, GXXYW may be effective in the suppression of the growth of endometriosis, possibly through the inhibition of cell proliferation, the induction of apoptosis of endometriotic cells, and the regulation of the immune system.

  1. Ganoderma tsugae Hepatoprotection against Exhaustive Exercise-Induced Liver Injury in Rats

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    Wan-Teng Lin

    2013-01-01

    Full Text Available Several studies have been shown that accelerated apoptosis is involved in post-exercise lymphocytopenia and tissue damage after high-intensity exercise. Ganoderma tsugae (GT is one of the well-known medicinal mushrooms that possess various pharmacological functions. This mushroom has traditionally been used for health promotion purposes. This study investigates the hepatoprotective effects of GT on exhaustive exercise-induced liver damage. Twenty-four male Sprague-Dawley rats were randomly divided into four groups and designated as exhaustive exercise only (E, exhaustive exercise with low dosage (EL, medium dosage (EM and high dosage (EH GT at 0, 0.1875, 0.9375 and 1.875 g/kg/day, respectively. After 30 days all rats were euthanized immediately after an exhaustive running challenge on a motorized treadmill. The rat livers were immediately harvested. Evidence of apoptotic liver cell death was revealed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay and caspases mediated cascade events. DNA fragmentation, an apoptosis process, can be examined using TUNEL assay. A few TUNEL-positive hepatocytes, compared to the exercise only group, were observed in the livers from exhaustive animals supplemented with GT. Immunoblot analysis also showed that caspase-6-mediated specific cleavage of lamin A/C was increased significantly in the livers of group E, but was significantly decreased in the EM and EH groups. Our observations demonstrate that GT possesses anti-apoptotic and hepatoprotective potential after exhaustive exercise.

  2. Ganoderma tsugae hepatoprotection against exhaustive exercise-induced liver injury in rats.

    Science.gov (United States)

    Huang, Chi-Chang; Huang, Wen-Ching; Yang, Suh-Ching; Chan, Chih-Chi; Lin, Wan-Teng

    2013-01-29

    Several studies have been shown that accelerated apoptosis is involved in post-exercise lymphocytopenia and tissue damage after high-intensity exercise. Ganoderma tsugae (GT) is one of the well-known medicinal mushrooms that possess various pharmacological functions. This mushroom has traditionally been used for health promotion purposes. This study investigates the hepatoprotective effects of GT on exhaustive exercise-induced liver damage. Twenty-four male Sprague-Dawley rats were randomly divided into four groups and designated as exhaustive exercise only (E), exhaustive exercise with low dosage (EL), medium dosage (EM) and high dosage (EH) GT at 0, 0.1875, 0.9375 and 1.875 g/kg/day, respectively. After 30 days all rats were euthanized immediately after an exhaustive running challenge on a motorized treadmill. The rat livers were immediately harvested. Evidence of apoptotic liver cell death was revealed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspases mediated cascade events. DNA fragmentation, an apoptosis process, can be examined using TUNEL assay. A few TUNEL-positive hepatocytes, compared to the exercise only group, were observed in the livers from exhaustive animals supplemented with GT. Immunoblot analysis also showed that caspase-6-mediated specific cleavage of lamin A/C was increased significantly in the livers of group E, but was significantly decreased in the EM and EH groups. Our observations demonstrate that GT possesses anti-apoptotic and hepatoprotective potential after exhaustive exercise.

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

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    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.

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

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    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.

  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. Substance P reduces apoptotic cell death possibly by modulating the immune response at the early stage after spinal cord injury.

    Science.gov (United States)

    Jiang, Mei Hua; Lim, Ji Eun; Chi, Guang Fan; Ahn, Woosung; Zhang, Mingzi; Chung, Eunkyung; Son, Youngsook

    2013-10-23

    Previously, we have reported that substance P (SP) enhanced functional recovery from spinal cord injury (SCI) possibly by the anti-inflammatory modulation associated with the induction of M2-type macrophages at the injured lesion. In this study, we explored the cytokine expression profiles and apoptotic cell death in the lesion site of the SCI after an immediate intravenous injection of SP. SP injection increased the levels of interleukin-4 (IL-4), IL-6, and IL-10 at day 1 after the SCI approximately by 2-, 9-, and 10-folds when compared with the control SCI, respectively. On the basis of double immunofluorescence staining with IL-10 and CD11b, activated macrophages or microglia expressing IL-10 appeared in the margin of the lesion site at day 1 only after the SP injection. This SP-mediated alteration in the lesion microenvironment was shown to be associated with the lower cell death of neuronal cells at day 1 and oligodendrocytes at day 5 by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which was also accompanied by a decrease in caspase-3 activation. These findings suggest that SP may reduce the inflammation-induced secondary cell death, possibly through immune modulation at an early stage after the SCI.

  7. A mitochondria-dependent pathway mediates the apoptosis of GSE-induced yeast.

    Science.gov (United States)

    Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun

    2012-01-01

    Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency.

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

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    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.

  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. 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.

  11. The expression of Smad4 after radiation of electromagnetic pulses and apoptosis in spermatogenic cells in mouse

    International Nuclear Information System (INIS)

    Ji Xinxin; Hou Wugang; Zhao Jie; Zhao Yong; Li Wei; Zhang Yuanqiang

    2007-01-01

    The aim of the study was to investigate the relationships between apoptosis induced by radiation of electromagnetic pulses (EMP) and the expression of Smad4 in mouse spermatogenic cells. 40 adult Balb/c mice were used, and 20 were irradiated with whole-body 400kV/m EMP. The mice were sacrificed and specimens were harvested at 1, 7, 14, 21 and 28 days after the irradiation. Histological changes were observed through Hematoxylin-Eosin staining (H-E staining), the apoptosis of spermatogenic cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL method) and the Smad4 expression was observed using immunohistochemistry SABC methods. Severe injuries were observed 1 day after the radiation and seminiferous epithelium was mostly recovered 28d after the radiation. The localization of smad4 was significantly different in EMP group compared to the control group, and the expression densities of smad4 decreased significantly at 7, 14 and 21d after irradiation (p<0.05). TUNEL assays demonstrated that there was a significant increase in the mean apoptotic index (AI) in irradiation groups than that of control groups (p<0.01). The results suggested that Smad4 and TGF-13/Smad signal pathway might play an important role in spermatogenic cells apoptosis induced by radiation of EMP. (authors)

  12. Protective effect of sulforaphane against retinal degeneration in the Pde6rd10 mouse model of retinitis pigmentosa.

    Science.gov (United States)

    Kang, Kai; Yu, Minzhong

    2017-12-01

    Retinitis pigmentosa (RP) is a group of inherited diseases characterized by the death of rod photoreceptors, followed by the death of cone photoreceptors, progressively leading to partial or complete blindness. Currently no specific treatment is available for RP patients. Sulforaphane (SFN) has been confirmed to be an effective antioxidant in the treatment of many diseases. In this study, we tested the therapeutic effects of SFN against photoreceptor degeneration in Pde6b rd10 mice. rd10 mice and C57/BL6 wild-type (WT) mice were treated with SFN and saline, respectively, from P6 to P20. Electroretinography (ERG), terminal deoxynucleotidyl transferase dUTP nick end labeling and western blot were tested, respectively, at P21 for the analysis of retinal function, retinal cell apoptosis or death and the protein express of GRP78/BiP (TUNEL) as a marker of endoplasmic reticulum (ER) stress. Compared with the saline group, the SFN-treated group showed significantly higher ERG a-wave and b-wave amplitudes, less photoreceptor death, and the downregulation of GRP78/BiP. Our data showed that SFN ameliorated the retinal degeneration of rd10 mice, which is possibly related to the downregulation of GRP78 expression.

  13. 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.

  14. Regulatory effect of neuroglobin in the recovery of spinal cord injury.

    Science.gov (United States)

    Dai, Ji-Lin; Lin, Yun; Yuan, Yong-Jian; Xing, Shi-Tong; Xu, Yi; Zhang, Qiang-Hua; Min, Ji-Kang

    2017-11-16

    The present study was aimed to investigate the therapeutic potential of neuroglobin in the recovery of spinal cord injury. The male albino Wistar strain rats were used as an experimental model, and adeno associated virus (AAV) was administered in the T12 section of spinal cord ten days prior to the injury. Basso Beattie Bresnahan (BBB) locomotor rating scale was used to determine the recovery of the hind limb during four weeks post-operation. Malondialdehyde (MDA), catalase and superoxide dismutase (SOD) were determined in the spinal cord tissues. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was carried out to determine the presence of apoptotic cells. Immunofluorescence analysis was carried out to determine the neuroglobin expression. Western blot analysis was carried out to determine the protein expressions of caspase-3, cytochrome c, bax and bcl-2 in the spinal cord tissues. Experimental results showed that rats were recovered from the spinal cord injury due to increased neuroglobin expression. Lipid peroxidation was reduced, whereas catalase and SOD activity were increased in the spinal cord tissues. Apoptosis and lesions were significantly reduced in the spinal cord tissues. Caspase-3, cytochrome c and bax levels were significantly reduced, whereas bcl-2 expression was reduced in the spinal cord tissues. Taking all these data together, it is suggested that the increased neuroglobin expression could improve the locomotor function.

  15. Valproic acid induces cutaneous wound healing in vivo and enhances keratinocyte motility.

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    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.

  16. 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.

  17. Branchial lesions associated with abundant apoptotic cells in oysters Ostrea edulis of Galicia (NW Spain).

    Science.gov (United States)

    Mirella da Silva, P; Villalba, Antonio; Sunila, Inke

    2006-06-12

    An experiment to evaluate differences in growth, mortality and disease susceptibility among Ostrea edulis stocks was performed. Five families were produced from each of 4 oyster populations (Irish, Greek and 2 Galician). The spat were transferred to a raft in the Ria de Arousa (Galicia, Spain) for grow-out. Monthly samples of each family were histologically processed from 2001 to 2003. One of the pathological conditions discovered by this study was the occurrence of extensive branchial lesions characterized by haemocytic infiltration and loss of branchial architecture. Furthermore, abundant atypical cells occurred among the haemocytes in the lesions in the branchial connective and epithelial tissues, but rarely in the mantle. These cells were contracted in size with nuclei showing chromatin condensation and fragmentation. Some nuclear chromatin aggregated under the nuclear membranes into crescent shapes, whereas others were uniformly dense. Those characteristics suggested that the cells were apoptotic haemocytes, which was confirmed by transmission electron microscopy (TEM) and by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay using the Apoptag Kit on paraffin sections. A low prevalence of gill lesions was detected in some, but not all, families of every origin peaking in July 2002 and April 2003. No etiologic agent was identified by either histology or TEM; thus, the cause of the abundance of apoptotic cells remains unclear.

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

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    Hideya Takahashi

    2014-04-01

    Full Text Available 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: Animals were euthanized after 5-10 d of fasting or feeding, or after 10-day fasting and 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: Plasma levels of cholesterol and glucose were reduced by fasting. Intestinal apoptosis 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. 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.

  19. Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway.

    Science.gov (United States)

    Ni, B; Bai, F F; Wei, Y; Liu, M J; Feng, Z X; Xiong, Q Y; Hua, L Z; Shao, G Q

    2015-09-25

    Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

  20. Effects of alpha particles on zebrafish embryos

    International Nuclear Information System (INIS)

    Yum, E.H.W.; Choi, V.W.Y.; Yu, K.N.; Li, V.W.T.; Cheng, S.H.

    2008-01-01

    Full text: Ionizing radiation such as X-ray and alpha particles can damage cellular macromolecules, which can lead to DNA single- and double-strand breaks. In the present work, we studied the effects of alpha particles on dechorionated zebrafish embryos. Thin polyallyldiglycol carbonate (PADC) films with a thickness of 16 μm were prepared from commercially available PADC films (with thickness of 100 μm) by chemical etching and used as support substrates for holding zebrafish embryos for alpha-particle irradiation. These films recorded alpha-particle hit positions, quantified the number and energy of alpha particles actually incident on the embryo cells, and thus enabled the calculation of the dose absorbed by the embryo cells. Irradiation was made at 1.25 hours post fertilization (hpf) with various absorbed dose. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed on the embryos at different time stages after irradiation. Marked apoptosis was detected only in embryos at earlier time stages. The results showed that DNA double-strand break during zebrafish embryogenesis can be induced by alpha-particle irradiation, which suggests that zebrafish is a potential model for assessing the effects of alpha-particle radiation

  1. Oxidative stress may be involved in distant organ failure in tourniquet shock model mice.

    Science.gov (United States)

    Nishikata, Rie; Kato, Naho; Hiraiwa, Kouichi

    2014-03-01

    Crush syndrome is characterized by prolonged shock resulting from extensive muscle damage and multiple organ failure. However, the pathogenesis of multiple organ failure has not yet been completely elucidated. Therefore, we investigated the molecular biological and histopathological aspects of distant organ injury in crush syndrome by using tourniquet shock model mice. DNA microarray analysis of the soleus muscle showed an increase in the mRNA levels of Cox-2, Hsp70, c-fos, and IL-6, at 3h after ischemia/reperfusion injury at the lower extremity. In vivo staining with hematoxylin and eosin (HE) showed edema and degeneration in the soleus muscle, but no change in the distant organs. Immunohistological staining of the HSP70 protein revealed nuclear translocation in the soleus muscle, kidney, liver, and lung. The c-fos mRNA levels were elevated in the soleus muscle, kidney, and liver, displaying nuclear translocation of c-FOS protein. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis suggested the involvement of apoptosis in ischemia/reperfusion injury in the soleus muscle. Apoptotic cells were not found in greater quantities in the kidney. Oxidative stress, as determined using a free radical elective evaluator (d-ROM test), markedly increased after ischemia/reperfusion injury. Therefore, examination of immunohistological changes and determination of oxidative stress are proposed to be useful in evaluating the extent of tourniquet shock, even before changes are observed by HE staining. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  2. Quercetin prevents pyrrolizidine alkaloid clivorine-induced liver injury in mice by elevating body defense capacity.

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    Lili Ji

    Full Text Available Quercetin is a plant-derived flavonoid that is widely distributed in nature. The present study is designed to analyze the underlying mechanism in the protection of quercetin against pyrrolizidine alkaloid clivorine-induced acute liver injury in vivo. Serum transaminases, total bilirubin analysis, and liver histological evaluation demonstrated the protection of quercetin against clivorine-induced liver injury. Terminal dUTP nick end-labeling assay demonstrated that quercetin reduced the increased amount of liver apoptotic cells induced by clivorine. Western-blot analysis of caspase-3 showed that quercetin inhibited the cleaved activation of caspase-3 induced by clivorine. Results also showed that quercetin reduced the increase in liver glutathione and lipid peroxidative product malondialdehyde induced by clivorine. Quercetin reduced the enhanced liver immunohistochemical staining for 4-hydroxynonenal induced by clivorine. Results of the Mouse Stress and Toxicity PathwayFinder RT2 Profiler PCR Array demonstrated that the expression of genes related with oxidative or metabolic stress and heat shock was obviously altered after quercetin treatment. Some of the alterations were confirmed by real-time PCR. Our results demonstrated that quercetin prevents clivorine-induced acute liver injury in vivo by inhibiting apoptotic cell death and ameliorating oxidative stress injury. This protection may be caused by the elevation of the body defense capacity induced by quercetin.

  3. Quercetin Prevents Pyrrolizidine Alkaloid Clivorine-Induced Liver Injury in Mice by Elevating Body Defense Capacity

    Science.gov (United States)

    Ji, Lili; Ma, Yibo; Wang, Zaiyong; Cai, Zhunxiu; Pang, Chun; Wang, Zhengtao

    2014-01-01

    Quercetin is a plant-derived flavonoid that is widely distributed in nature. The present study is designed to analyze the underlying mechanism in the protection of quercetin against pyrrolizidine alkaloid clivorine-induced acute liver injury in vivo. Serum transaminases, total bilirubin analysis, and liver histological evaluation demonstrated the protection of quercetin against clivorine-induced liver injury. Terminal dUTP nick end-labeling assay demonstrated that quercetin reduced the increased amount of liver apoptotic cells induced by clivorine. Western-blot analysis of caspase-3 showed that quercetin inhibited the cleaved activation of caspase-3 induced by clivorine. Results also showed that quercetin reduced the increase in liver glutathione and lipid peroxidative product malondialdehyde induced by clivorine. Quercetin reduced the enhanced liver immunohistochemical staining for 4-hydroxynonenal induced by clivorine. Results of the Mouse Stress and Toxicity PathwayFinder RT2 Profiler PCR Array demonstrated that the expression of genes related with oxidative or metabolic stress and heat shock was obviously altered after quercetin treatment. Some of the alterations were confirmed by real-time PCR. Our results demonstrated that quercetin prevents clivorine-induced acute liver injury in vivo by inhibiting apoptotic cell death and ameliorating oxidative stress injury. This protection may be caused by the elevation of the body defense capacity induced by quercetin. PMID:24905073

  4. Hataedock treatment has preventive therapeutic effects for atopic dermatitis through skin barrier protection in Dermatophagoides farinae-induced NC/Nga mice.

    Science.gov (United States)

    Cha, Ho-Yeol; Ahn, Sang-Hyun; Cheon, Jin-Hong; Park, Sun-Young; Kim, Kibong

    2017-07-12

    Hataedock treatment is traditionally used for the purpose of preventing the future skin disease by feeding herbal extracts to the newborn in traditional Chinese and Korean medicine. This study investigated the preventive therapeutic effects of Hataedock (HTD) treatment for atopic dermatitis (AD) through skin barrier protection in Dermatophagoides farinae-induced NC/Nga mice. To the HTD treatment group, the extract of Coptis japonica Makino and Glycyrrhiza uralensis Fischer, which analyzed with High Performance Liquid Chromatography (HPLC)-fingerprint for quality consistency, was administered orally to the 3-week-old mice before inducing AD. After that, Dermatophagoides farinae was applied except the control group to induce AD-like skin lesions. We confirmed the effects of HTD on morphological changes, protection of skin barrier, regulation of Th2 differentiation, inflammation regulation and induction of apoptosis through histochemistry, immunohistochemistry, and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. HTD effectively reduced edema, angiogenesis and skin lesion. HTD also increased the levels of liver X receptor (LXR) and filaggrin but decreased the level of protein kinase C (PKC) (pprotection of skin barrier. Therefore, HTD may have potential applications for alternative and preventive treatment in the management of AD. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  5. Radiation-induced hyperproliferation of intestinal crypts results in elevated genome instability with inactive p53-related genomic surveillance.

    Science.gov (United States)

    Zhou, Xin; Ma, Xiaofei; Wang, Zhenhua; Sun, Chao; Wang, Yupei; He, Yang; Zhang, Hong

    2015-12-15

    Radiation-induced hyperproliferation of intestinal crypts is well documented, but its potential tumorigenic effects remain elusive. Here we aim to determine the genomic surveillance process during crypt hyperproliferation, and its consequential outcome after ionizing radiation. Crypt regeneration in the intestine was induced by a single dose of 12Gy abdominal irradiation. γ-H2AX, 53BP1 and DNA-PKcs were used as DNA repair surrogates to investigate the inherent ability of intestinal crypt cells to recognize and repair double-strand breaks. Ki67 staining and the 5-bromo-2'-deoxyuridine incorporation assay were used to study patterns of cell proliferation in regenerating crypts. Staining for ATM, p53, Chk1 and Chk2 was performed to study checkpoint activation and release. Apoptosis was evaluated through H&E staining and terminal deoxynucleotidyl transferase (dUTP) nick-end labeling. The ATM-p53 pathway was immediately activated after irradiation. A second wave of DSBs in crypt cells was observed in regenerating crypts, accompanied with significantly increased chromosomal bridges. The p53-related genomic surveillance pathway was not active during the regeneration phase despite DSBs and chromosomal bridges in the cells of regenerating crypts. Non-homologous end joining (NHEJ) DSBs repair was involved in the DSBs repair process, as indicated by p-DNA-PKcs staining. Intestinal crypt cells retained hyperproliferation with inactive p53-related genomic surveillance system. NHEJ was involved in the resultant genomic instability during hyperproliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Shaofu Zhuyu Decoction Regresses Endometriotic Lesions in a Rat Model

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    Guanghui Zhu

    2018-01-01

    Full Text Available The current therapies for endometriosis are restricted by various side effects and treatment outcome has been less than satisfactory. Shaofu Zhuyu Decoction (SZD, a classic traditional Chinese medicinal (TCM prescription for dysmenorrhea, has been widely used in clinical practice by TCM doctors to relieve symptoms of endometriosis. The present study aimed to investigate the effects of SZD on a rat model of endometriosis. Forty-eight female Sprague-Dawley rats with regular estrous cycles went through autotransplantation operation to establish endometriosis model. Then 38 rats with successful ectopic implants were randomized into two groups: vehicle- and SZD-treated groups. The latter were administered SZD through oral gavage for 4 weeks. By the end of the treatment period, the volume of the endometriotic lesions was measured, the histopathological properties of the ectopic endometrium were evaluated, and levels of proliferating cell nuclear antigen (PCNA, CD34, and hypoxia inducible factor- (HIF- 1α in the ectopic endometrium were detected with immunohistochemistry. Furthermore, apoptosis was assessed using the terminal deoxynucleotidyl transferase (TdT deoxyuridine 5′-triphosphate (dUTP nick-end labeling (TUNEL assay. In this study, SZD significantly reduced the size of ectopic lesions in rats with endometriosis, inhibited cell proliferation, increased cell apoptosis, and reduced microvessel density and HIF-1α expression. It suggested that SZD could be an effective therapy for the treatment and prevention of endometriosis recurrence.

  7. Epidermal cell death in frogs with chytridiomycosis

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    Laura A. Brannelly

    2017-02-01

    Full Text Available 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.

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

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    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.

  9. Human scalp hair follicles are both a target and a source of prolactin, which serves as an autocrine and/or paracrine promoter of apoptosis-driven hair follicle regression.

    Science.gov (United States)

    Foitzik, Kerstin; Krause, Karoline; Conrad, Franziska; Nakamura, Motonobu; Funk, Wolfang; Paus, Ralf

    2006-03-01

    The prototypic pituitary hormone prolactin (PRL) exerts a wide variety of bioregulatory effects in mammals and is also found in extrapituitary sites, including murine skin. Here, we show by reverse transcriptase-polymerase chain reaction and immunohistology that, contrary to a previous report, human skin and normal human scalp hair follicles (HFs), in particular, express both PRL and PRL receptors (PRL-R) at the mRNA and protein level. PRL and PRL-R immunoreactivity can be detected in the epithelium of human anagen VI HFs, while the HF mesenchyme is negative. During the HF transformation from growth (anagen) to apoptosis-driven regression (catagen), PRL and PRL-R immunoreactivity appear up-regulated. Treatment of organ-cultured human scalp HFs with high-dose PRL (400 ng/ml) results in a significant inhibition of hair shaft elongation and premature catagen development, along with reduced proliferation and increased apoptosis of hair bulb keratinocytes (Ki-67/terminal dUTP nick-end labeling immunohistomorphometry). This shows that PRL receptors, expressed in HFs, are functional and that human skin and human scalp HFs are both direct targets and sources of PRL. Our data suggest that PRL acts as an autocrine hair growth modulator with catagen-promoting functions and that the hair growth-inhibitory effects of PRL demonstrated here may underlie the as yet ill-understood hair loss in patients with hyper-prolactinemia.

  10. 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.

  11. Total flavonoid extract from Coreopsis tinctoria Nutt. protects rats against myocardial ischemia/reperfusion injury.

    Science.gov (United States)

    Zhang, Ya; Yuan, Changsheng; Fang, He; Li, Jia; Su, Shanshan; Chen, Wen

    2016-09-01

    This study aimed to evaluate the protective effects of total flavonoid extract from Coreopsis tinctoria Nutt. (CTF) against myocardial ischemia/reperfusion injury (MIRI) using an isolated Langendorff rat heart model. Left ventricular developed pressure (LVDP) and the maximum rate of rise and fall of LV pressure (±dp/dtmax) were recorded. Cardiac injury was assessed by analyzing lactate dehydrogenase (LDH) and creatine kinase (CK) released in the coronary effluent. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels were determined. Myocardial inflammation was assessed by monitoring tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), interleukin-8 (IL-8), and interleukin-6 (IL-6) levels. Myocardial infarct size was estimated. Cell morphology was assessed by 2,3,5-triphenyltetrazolium chloride and hematoxylin and eosin (HE) staining. Cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Pretreatment with CTF significantly increased the heart rate and increased LVDP, as well as SOD and GSH-Px levels. In addition, CTF pretreatment decreased the TUNEL-positive cell ratio, infarct size, and levels of CK, LDH, MDA, TNF-α, CRP, IL-6, and IL-8. These results suggest that CTF exerts cardio-protective effects against MIRI via anti-oxidant, anti-inflammatory, and anti-apoptotic activities.

  12. Resveratrol Enhances Neurite Outgrowth and Synaptogenesis Via Sonic Hedgehog Signaling Following Oxygen-Glucose Deprivation/Reoxygenation Injury

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    Fanren Tang

    2017-09-01

    Full Text Available Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R. Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo agonist purmophamine, which activates Shh signaling, increased viability, reduced apoptosis, and stimulated neurite outgrowth after OGD/R injury. Moreover, the expression of growth-associated protein(GAP-43, synaptophysin, Shh, Patched (Ptc-1, Smo, glioma-associated oncogene homolog (Gli-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1.

  13. 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.

  14. Nerve growth factor reduces apoptotic cell death in rat facial motor neurons after facial nerve injury.

    Science.gov (United States)

    Hui, Lian; Yuan, Jing; Ren, Zhong; Jiang, Xuejun

    2015-01-01

    To assess the effects of nerve growth factor (NGF) on motor neurons after induction of a facial nerve lesion, and to compare the effects of different routes of NGF injection on motor neuron survival. This study was carried out in the Department of Otolaryngology Head & Neck Surgery, China Medical University, Liaoning, China from October 2012 to March 2013. Male Wistar rats (n = 65) were randomly assigned into 4 groups: A) healthy controls; B) facial nerve lesion model + normal saline injection; C) facial nerve lesion model + NGF injection through the stylomastoid foramen; D) facial nerve lesion model + intraperitoneal injection of NGF. Apoptotic cell death was detected using the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Expression of caspase-3 and p53 up-regulated modulator of apoptosis (PUMA) was determined by immunohistochemistry. Injection of NGF significantly reduced cell apoptosis, and also greatly decreased caspase-3 and PUMA expression in injured motor neurons. Group C exhibited better efficacy for preventing cellular apoptosis and decreasing caspase-3 and PUMA expression compared with group D (pfacial nerve injury in rats. The NGF injected through the stylomastoid foramen demonstrated better protective efficacy than when injected intraperitoneally.

  15. 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

  16. 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.

  17. 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

  18. Adresát Jindřich Honzl. Dopisy a scénické návrhy Jindřicha Štyrského z Honzlovy pozůstalosti

    Czech Academy of Sciences Publication Activity Database

    Bydžovská, Lenka; Srp, K.

    2008-01-01

    Roč. 56, č. 6 (2008), s. 529-537 ISSN 0049-5123 R&D Projects: GA AV ČR IAA800330603 Institutional research plan: CEZ:AV0Z80330511 Keywords : stage design * artificialism * surrealism Subject RIV: AL - Art, Architecture, Cultural Heritage

  19. 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

  20. 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

  1. 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

    Directory of Open Access Journals (Sweden)

    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

  2. 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

    Directory of Open Access Journals (Sweden)

    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

  3. 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

    Directory of Open Access Journals (Sweden)

    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

  4. Recombinant adenovirus-mediated overexpression of PTEN and KRT10 improves cisplatin resistance of ovarian cancer in vitro and in vivo.

    Science.gov (United States)

    Wu, H; Wang, K; Liu, W; Hao, Q

    2015-06-18

    Drug resistance is a major cause of treatment failure in ovarian cancer patients, and novel therapeutic strategies are urgently needed. Overexpression of phosphatase and tensin homolog (PTEN) has been shown to preserve the cisplatin-resistance of ovarian cancer cells, while cisplatin-induced keratin 10 (KRT10) overexpression mediates the resistance-reversing effect of PTEN. However, whether overexpression of PTEN or KRT10 can improve the cisplatin resistance of ovarian cancer in vivo has not been investigated. Therefore, we investigated the effects of adenovirus-mediated PTEN or KRT10 overexpression on the cisplatin resistance of ovarian cancer in vivo. Recombinant adenoviruses carrying the gene for PTEN or KRT10 were constructed. The effects of overexpression of PTEN and KRT10 on cisplatin resistance of ovarian cancer cells were examined using the 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and TdT-mediated dUTP nick-end labeling (TUNEL) assays in vitro. Subcutaneously transplanted nude mice, as a model of human ovarian cancer, were used to test the effects of PTEN and KRT10 on cisplatin resistance of ovarian cancer in vivo. The MTT assay showed that recombinant adenovirus-mediated overexpression of KRT10 and PTEN enhanced the proliferation inhibition effect of cisplatin on C13K cells. Recombinant adenovirus-mediated overexpression of KRT10 and PTEN also increased the cisplatin-induced apoptosis rate of C13K cells. Furthermore, recombinant adenovirus-mediated overexpression of KRT10 and PTEN enhanced the inhibitory effect of cisplatin on C13K xenograft tumor growth. Thus, recombinant adenovirus-mediated overexpression of KRT10 and PTEN may improve the cisplatin resistance of ovarian cancer in vitro and in vivo.

  5. Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.

    Science.gov (United States)

    Ruwanpura, Saleela M; McLeod, Louise; Dousha, Lovisa F; Seow, Huei J; Alhayyani, Sultan; Tate, Michelle D; Deswaerte, Virginie; Brooks, Gavin D; Bozinovski, Steven; MacDonald, Martin; Garbers, Christoph; King, Paul T; Bardin, Philip G; Vlahos, Ross; Rose-John, Stefan; Anderson, Gary P; Jenkins, Brendan J

    2016-12-15

    The potent immunomodulatory cytokine IL-6 is consistently up-regulated in human lungs with emphysema and in mouse emphysema models; however, the mechanisms by which IL-6 promotes emphysema remain obscure. IL-6 signals using two distinct modes: classical signaling via its membrane-bound IL-6 receptor (IL-6R), and trans-signaling via a naturally occurring soluble IL-6R. To identify whether IL-6 trans-signaling and/or classical signaling contribute to the pathogenesis of emphysema. We used the gp130 F/F genetic mouse model for spontaneous emphysema and cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various methods including in vivo lung function and stereology, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and location of IL-6 signaling-related genes and proteins were measured, and the levels of IL-6 and related proteins in sera from emphysematous mice and patients were also assessed. Lung tissues from patients with emphysema, and from spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by excessive production of soluble IL-6R. Genetic blockade of IL-6 trans-signaling in emphysema mouse models and therapy with the IL-6 trans-signaling antagonist sgp130Fc ameliorated emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6 trans-signaling-driven emphysematous changes in the lung correlated with mechanistic target of rapamycin complex 1 hyperactivation, and treatment of emphysema mouse models with the mechanistic target of rapamycin complex 1 inhibitor rapamycin attenuated emphysematous changes. Collectively, our data reveal that specific targeting of IL-6 trans-signaling may represent a novel treatment strategy for emphysema.

  6. Overexpression of interleukin-1β and interferon-γ in type I thoracic aortic dissections and ascending thoracic aortic aneurysms: possible correlation with matrix metalloproteinase-9 expression and apoptosis of aortic media cells.

    Science.gov (United States)

    Zhang, Lei; Liao, Ming-fang; Tian, Lei; Zou, Si-li; Lu, Qing-sheng; Bao, Jun-min; Pei, Yi-fei; Jing, Zai-ping

    2011-07-01

    To examine the expression of interleukin-1β and interferon-γ and their possible roles in aortic dissections and aneurysms. Aortic specimens were obtained from patients with type I thoracic aortic dissection, ascending thoracic aortic aneurysms, and control organ donors. The expression of interleukin-1β, interferon-γ, matrix metalloproteinase-9, and signal transduction factors phospho-p38 and phosphorylated c-jun N-terminal kinase (phospho-JNK) were detected by real time reverse transcription-polymerase chain reaction (real time RT-PCR), Western blot, and immunohistochemistry, respectively. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed to detect apoptosis of media cells. The correlation of these factors and apoptosis was also studied. Apoptosis in the media of thoracic aortic dissection and in ascending thoracic aortic aneurysms was dramatically higher than in the control group. The expression of interleukin-1β gradually increased from the control group, thoracic aortic dissection to ascending thoracic aortic aneurysms (p matrix metalloproteinase-9 was significantly increased in the media of thoracic aortic dissection and ascending thoracic aortic aneurysms compared with the control group (p correlations between interleukin-1β versus matrix metalloproteinase-9, interleukin-1β versus phospho-p38 in thoracic aortic dissection (p matrix metalloproteinase-9, interferon-γ versus phospho-JNK, interferon-γ versus apoptosis, and interleukin-1β versus apoptosis in ascending thoracic aortic aneurysms (p = 0.02, 0.02, p matrix metalloproteinase-9 and the apoptosis of media cells in humans. Copyright © 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

  7. Classical swine fever virus induces pyroptosis in the peripheral lymphoid organs of infected pigs.

    Science.gov (United States)

    Yuan, Jin; Zhu, Mengjiao; Deng, Shaofeng; Fan, Shuangqi; Xu, Hailuan; Liao, Jiedan; Li, Peng; Zheng, Jingfang; Zhao, Mingqiu; Chen, Jinding

    2018-05-02

    Classical swine fever virus (CSFV) causes a highly lethal disease in pigs, which is characterized by immunosuppression. Leukopenia is known to be a possible mechanism of immunosuppression during CSFV infection. As a new and specialized form of cell death, pyroptosis is the key response of the innate immune system to pathogens, and is widely involved in the occurrence and development of infectious diseases. However, the relationship between CSFV and pyroptosis has not been explored. In this study, we investigated the occurrence of pyroptosis in pigs following CSFV infection. According to qRT-PCR assay results, the prevalence of this virus in peripheral lymphoid organs (tonsils, lymph nodes, and spleen) was much higher than that in other organs. Severe bleeding, necrosis, and a significant reduction in lymphocytes were found in the peripheral lymphoid organs of CSFV-infected pigs based on histological examination. In-depth studies showed that an increased ratio of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were present in the peripheral lymphoid organs of the CSFV-infected group according to immunohistochemistry. Meanwhile, the p10 subunit and activity of caspase-1, which is a regulator of pyroptosis, the N-terminal domain of gasdermin D, which is an executor of pyroptosis, and the cleavage and secretion of IL-1b, which is a product of pyroptosis were increased in the peripheral lymphoid organs of the CSFV-infected group. Together, these results demonstrated that pyroptosis is involved in CSFV-induced cell death in vivo, which provides a new understanding of the mechanism associated with lymphocyte depletion and immunosuppression in pigs infected with this virus. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. 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

  9. Gualou Xiebai Decoction, a Traditional Chinese Medicine, Prevents Cardiac Reperfusion Injury of Hyperlipidemia Rat via Energy Modulation.

    Science.gov (United States)

    Yan, Lu-Lu; Zhang, Wei-Yang; Wei, Xiao-Hong; Yan, Li; Pan, Chun-Shui; Yu, Yang; Fan, Jing-Yu; Liu, Yu-Ying; Zhou, Hua; Han, Jing-Yan; Yao, Xin-Sheng

    2018-01-01

    Background: Gualou Xiebai Decoction (GLXB) is a classic prescription of Chinese medicine used for the treatment of cardiac problems. The present study was designed to explore the effect and mechanism of GLXB on ischemia/reperfusion (I/R) induced disorders in myocardial structure and function, focusing on the regulation of energy metabolism and the RhoA/ROCK pathway. Methods: After hyperlipidemic rat model was established by oral administration of high fat diet, the rats were treated with GLXB for 6 weeks and subjected to 30 min occlusion of the left anterior descending coronary artery (LADCA) followed by 90 min reperfusion to elicit I/R challenge. Myocardial infarct size was assessed by Evans blue-TTC staining. Myocardial blood flow (MBF) and cardiac function were evaluated. Enzyme-linked immunosorbent assay was performed to examine the content of ATP, ADP, AMP, CK, CK-MB, LDH, cTnT, cTnI, and IL-6. Double staining of F-actin and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was conducted to assess myocardial apoptosis. Expressions of ATP synthase subunit δ (ATP 5D), and RhoA and ROCK were determined by Western blotting. Results: Administration with GLXB at high dose for 6 weeks protected heart against I/R-induced MBF decrease, myocardial infarction and apoptosis, ameliorated I/R-caused impairment of cardiac function and myocardial structure, restored the decrease in the ratio of ADP/ATP and AMP/ATP, and the expression of ATP 5D with inhibiting the expression of RhoA and ROCK. Conclusions: Treatment with GLXB effectively protects myocardial structure and function from I/R challenge, possibly via regulating energy metabolism involving inactivation of RhoA/ROCK signaling pathway.

  10. Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardial ischemia/reperfusion injury in rats.

    Science.gov (United States)

    Jian, Jie; Xuan, Feifei; Qin, Feizhang; Huang, Renbin

    2015-01-01

    This study aimed to determine the effects of Bauhinia championii flavone (BCF) on myocardial ischemia/reperfusion injury (MI/RI) in rats and to explore potential mechanisms. The MI/RI model in rats was established by ligating the left anterior descending coronary artery for 30 minutes, then reperfusing for 3 hours. BCF at 20 mg/kg was given 20 minutes prior to ischemia via sublingual intravenous injection, with 24 μg/kg phosphoinositide 3-kinase inhibitor (PI3K; wortmannin) as a control. The creatine kinase-MB and nitric oxide content were assessed by colorimetry. The levels of mitochondrial permeability transition pores and tumor necrosis factor alpha were determined by an enzyme-linked immunosorbent assay. Cardiomyocyte apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the expression of PI3K, endothelial nitric oxide synthase, caspase-3, and Beclin1 was analyzed by fluorescence quantitative polymerase chain reaction and Western blotting, respectively. Akt and microtubule-associated protein 1 light chain 3-II protein levels were also evaluated. Pretreatment with BCF significantly decreased the levels of creatine kinase-MB, tumor necrosis factor alpha, and mitochondrial permeability transition pores, but increased the nitric oxide content. Furthermore, BCF inhibited apoptosis, downregulated caspase-3, Beclin1, and microtubule-associated protein 1 light chain 3-II, upregulated PI3K, and increased the protein levels of phosphorylated Akt and endothelial nitric oxide synthase. However, all of the previously mentioned effects of BCF were blocked when BCF was coadministered with wortmannin. In conclusion, these observations indicated that BCF has cardioprotective effects against MI/RI by reducing cell apoptosis and excessive autophagy, which might be related to the activation of the PI3K/Akt signaling pathway.

  11. The Antioxidant, Anti-Inflammatory and Anti-Apoptotic Activities of the Bauhinia Championii Flavone are Connected with Protection Against Myocardial Ischemia/Reperfusion Injury.

    Science.gov (United States)

    Jian, Jie; Xuan, Feifei; Qin, Feizhang; Huang, Renbin

    2016-01-01

    Previous studies have demonstrated that Bauhinia championii flavone (BCF) exhibits anti-oxidative, anti-hypoxic and anti-stress properties. This study was designed to investigate whether BCF has a cardioprotective effect against myocardial ischemia/reperfusion (I/R) injuries in rats and to shed light on its possible mechanism. The model of I/R was established by ligating the left anterior descending coronary artery for 30 min, then reperfusing for 180 min. Hemodynamic changes were continuously monitored. The content of malondialdehyde (MDA) as well as the lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were assessed. The release of interleukin-6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA). Apoptosis of cardiomyocytes was determined by caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of TLR4, NF-x03BA;Bp65, Bcl-2 and Bax were detected by western blotting. Pretreatment with BCF significantly reduced the serum levels of LDH, MDA and IL-6, but increased the activities of SOD and GSH-Px. It also attenuated myocardial infarct size, reduced the apoptosis rate and preserved cardiac function. Furthermore, BCF inhibited caspase-3 activity and the expression of TLR4, phosphorylated NF-x03BA;Bp65 and Bax, but enhanced the expression of Bcl-2. These results provide substantial evidence that BCF exerts a protective effect on myocardial I/R injury, which may be attributed to attenuating lipid peroxidation, the inflammatory response and apoptosis. © 2016 The Author(s) Published by S. Karger AG, Basel.

  12. Apoptotic mechanisms after repeated noise trauma in the mouse medial geniculate body and primary auditory cortex.

    Science.gov (United States)

    Fröhlich, Felix; Ernst, Arne; Strübing, Ira; Basta, Dietmar; Gröschel, Moritz

    2017-12-01

    A correlation between noise-induced apoptosis and cell loss has previously been shown after a single noise exposure in the cochlear nucleus, inferior colliculus, medial geniculate body (MGB) and primary auditory cortex (AI). However, repeated noise exposure is the most common situation in humans and a major risk factor for the induction of noise-induced hearing loss (NIHL). The present investigation measured cell death pathways using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in the dorsal, medial and ventral MGB (dMGB, mMGB and vMGB) and six layers of the AI (AI-1 to AI-6) in mice (NMRI strain) after a second noise exposure (double-exposure group). Therefore, a single noise exposure group has been investigated 7 (7-day-group-single) or 14 days (14-day-group-single) after noise exposure (3 h, 5-20 kHz, 115 dB SPL peak-to-peak). The double-exposure group received the same noise trauma for a second time 7 days after the initial exposure and was either TUNEL-stained immediately (7-day-group-double) or 1 week later (14-day-group-double) and data were compared to the corresponding single-trauma group as well as to an unexposed control group. It was shown that TUNEL increased immediately after the second noise exposure in AI-3 and stayed upregulated in the 14-day-group-double. A significant increase in TUNEL was also seen in the 14-day-group-double in vMGB, mMGB and AI-1. The present results show for the first time the influence of a repeated noise trauma on cell death mechanisms in thalamic and cortical structures and might contribute to the understanding of pathophysiological findings and psychoacoustic phenomena accompanying NIHL.

  13. Activated Α7nachr Improves Postoperative Cognitive Dysfunction and Intestinal Injury Induced by Cardiopulmonary Bypass in Rats: Inhibition of the Proinflammatory Response Through the Th17 Immune Response

    Directory of Open Access Journals (Sweden)

    Keyan Chen

    2018-04-01

    Full Text Available Backgrund/Aims: To investigate the effects of activated α7 nicotinic acetylcholine receptor (α7nAChR on postoperative cognitive dysfunction (POCD and intestinal injury induced by cardiopulmonary bypass (CPB and its relationship with the Th17 response in order to provide a theoretical basis for organ protection and targeted drug therapy during the perioperative period. Methods: Sprague-Dawley rat models of CPB were established. Rat intestinal and brain injuries were observed after CPB using hematoxylin and eosin staining. Cell apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling. Inflammatory factors and markers of brain injury in rat serum were measured using enzyme-linked immunosorbent assay. The expression levels of Bcl-2, Bax, caspase-3, ZO-1, occludin, AQP4, RORγT, and α7nAchR were examined using western blotting. Transcription factor RORγT expression was determined using real-time fluorescent quantitative polymerase chain reaction. Th17 cells in the peripheral blood and spleen were determined using flow cytometry. α7nAchR knockout rats were established. The Th17 response in the peripheral blood and spleen of α7nAchR knockout rats was further verified using flow cytometry. Results: CPB can induce POCD and intestinal injury in rats. α7nAchR activation markedly reduced intestinal injury, POCD, neuronal apoptosis, proinflammatory factor expression, and number of CD4+IL-17+ cells. α7nAchR knockout significantly increased serum D-lactic acid, FABP2, S-100β, NSE, TNF-α, IL-6, and IL-17 secretion. The number of CD4+IL-17+ cells was also significantly increased. Conclusion: α7nAchR activation markedly ameliorates the intestinal injury and POCD induced by CPB. Inhibition of the Th17 immune response can reduce the proinflammatory response, which could provide a new method for clinical perioperative organ protection and targeted drug therapy.

  14. Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience.

    Science.gov (United States)

    Toker, M Berk; Alcay, Selim; Gokce, Elif; Ustuner, Burcu

    2016-06-01

    The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3-5 on a 0-5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups 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). Semen samples also incubated for 6 h in humidified air with 5% CO2 at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Liver atrophy after percutaneous transhepatic portal embolization occurs in two histological phases: Hepatocellular atrophy followed by apoptosis.

    Science.gov (United States)

    Iwao, Yasuhito; Ojima, Hidenori; Kobayashi, Tatsushi; Kishi, Yoji; Nara, Satoshi; Esaki, Minoru; Shimada, Kazuaki; Hiraoka, Nobuyoshi; Tanabe, Minoru; Kanai, Yae

    2017-11-18

    To clarify the histological changes associated with liver atrophy after percutaneous transhepatic portal embolization (PTPE) in pigs and humans. As a preliminary study, we performed pathological examinations of liver specimens from five pigs that had undergone PTPE in a time-dependent model of liver atrophy. In specimens from embolized lobes (EMB) and nonembolized lobes (controls), we measured the portal vein to central vein distance (PV-CV), the area and number of hepatocytes per lobule, and apoptotic activity using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Immunohistochemical reactivities were evaluated for light chain 3 (LC3) and lysosomal-associated membrane protein 2 (LAMP2) as autophagy markers and for glutamine synthetase and cytochrome P450 2E1 (CYP2E1) as metabolic zonation markers. Samples from ten human livers taken 20-36 d after PTPE were similarly examined. PV-CVs and lobule areas did not differ between EMB and controls at day 0, but were lower in EMB than in controls at weeks 2, 4, and 6 ( P ≤ 0.001). Hepatocyte numbers were not significantly reduced in EMB at day 0 and week 2 but were reduced at weeks 4 and 6 ( P ≤ 0.05). Apoptotic activity was higher in EMB than in controls at day 0 and week 4. LC3 and LAMP2 staining peaked in EMB at week 2, with no significant difference between EMB and controls at weeks 4 and 6. Glutamine synthetase and CYP2E1 zonation in EMB at weeks 2, 4, and 6 were narrower than those in controls. Human results were consistent with those of porcine specimens. The mechanism of liver atrophy after PTPE has two histological phases: Hepatocellular atrophy is likely caused by autophagy in the first 2 wk and apoptosis thereafter.

  16. Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

    Directory of Open Access Journals (Sweden)

    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

  17. 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.

  18. Differential expression and role of hyperglycemia induced oxidative stress in epigenetic regulation of β1, β2 and β3-adrenergic receptors in retinal endothelial cells

    Science.gov (United States)

    2014-01-01

    Background Aberrant epigenetic profiles are concomitant with a spectrum of developmental defects and diseases. Role of methylation is an increasingly accepted factor in the pathophysiology of diabetes and its associated complications. This study aims to examine the correlation between oxidative stress and methylation of β1, β2 and β3-adrenergic receptors and to analyze the differential variability in the expression of these genes under hyperglycemic conditions. Methods Human retinal endothelial cells were cultured in CSC complete medium in normal (5 mM) or high (25 mM) glucose to mimic a diabetic condition. Reverse transcription PCR and Western Blotting were performed to examine the expression of β1, β2 and β3-adrenergic receptors. For detections, immunocytochemistry was used. Bisulfite sequencing method was used for promoter methylation analysis. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to measure reactive oxygen species (ROS) production in the cells. Results β1 and β3-adrenergic receptors were expressed in retinal endothelial cells while β2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on β1 and β2-adrenergic receptors methylation and expression however β3-adrenergic receptors showed a significantly higher expression (p adrenergic receptors methylation with no significant effect on β1 and β2-adrenergic receptors. β2-adrenergic receptor was hypermethylated with halted expression. Conclusion Our study demonstrates that β1 and β3-adrenergic receptors expressed in human retinal endothelial cells. Oxidative stress and apoptosis are inversely proportional to the extent of promoter methylation, suggesting that methylation loss might be due to oxidative stress-induced DNA damage. PMID:24885710

  19. Palmitate induces VSMC apoptosis via toll like receptor (TLR)4/ROS/p53 pathway.

    Science.gov (United States)

    Zhang, Yuanjun; Xia, Guanghao; Zhang, Yaqiong; Liu, Juxiang; Liu, Xiaowei; Li, Weihua; Lv, Yaya; Wei, Suhong; Liu, Jing; Quan, Jinxing

    2017-08-01

    Toll-like receptor 4 (TLR4) has been implicated in vascular inflammation, as well as in the pathogenesis of atherosclerosis and diabetes. Vascular smooth muscle cell (VSMC) apoptosis has been shown to induce plaque vulnerability in atherosclerosis. Previous studies reported that palmitate induced apoptosis in VSMCs; however, the role of TLR4 in palmitate-induced apoptosis in VSMCs has not yet been defined. In this study, we investigated whether or not palmitate-induced apoptosis depended on the activation of the TLR4 pathway. VSMCs were treated with or without palmitate, CRISPR/Cas9z-mediated genome editing methods were used to deplete TLR4 expression, while NADPH oxidase inhibitors were used to inhibit reactive oxygen species (ROS) generation. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, ROS was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method, the mRNA and protein expression levels of caspase 3, caspase 9, BCL-2 and p53 were studied by real-time polymerase chain reaction (RT-PCR) and ELISA. Palmitate significantly promotes VSMC apoptosis, ROS generation, and expression of caspase 3, caspase 9 and p53; while NADPH oxidase inhibitor pretreatment markedly attenuated these effects. Moreover, knockdown of TLR4 significantly blocked palmitate-induced ROS generation and VSMC apoptosis accompanied by inhibition of caspase 3, caspase 9, p53 expression and restoration of BCL-2 expression. Our results suggest that palmitate-induced apoptosis depends on the activation of the TLR4/ROS/p53 signaling pathway, and that TLR4 may be a potential therapeutic target for the prevention and treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Bmi-1-targeting suppresses osteosarcoma aggressiveness through the NF-κB signaling pathway

    Science.gov (United States)

    Liu, Jiaguo; Luo, Bin; Zhao, Meng

    2017-01-01

    Bone cancer is one of the most lethal malignancies and the specific causes of tumor initiation are not well understood. B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been reported to be associated with the initiation and progression of osteosarcoma, and as a prognostic indicator in the clinic. In the current study, a full-length antibody targeting Bmi-1 (AbBmi-1) was produced and the preclinical value of Bmi-1-targeted therapy was evaluated in bone carcinoma cells and tumor xenograft mice. The results indicated that the Bmi-1 expression level was markedly upregulated in bone cancer cell lines, and inhibition of Bmi-1 by AbBmi-1 reduced the invasiveness and migration of osteosarcoma cells. Overexpression of Bmi-1 promoted proliferation and angiogenesis, and increased apoptosis resistance induced by cisplatin via the nuclear factor-κB (NF-κB) signal pathway. In addition, AbBmi-1 treatment inhibited the tumorigenicity of osteosarcoma cells in vivo. Furthermore, AbBmi-1 blocked NF-κB signaling and reduced MMP-9 expression. Furthermore, Bmi-1 promoted osteosarcoma tumor growth, whereas AbBmi-1 significantly inhibited osteosarcoma tumor growth in vitro and in vivo. Notably, AbBmi-1 decreased the percentages of Ki67-positive cells and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in tumors compared with Bmi-1-treated and PBS controls. Notably, MMP-9 and NF-κB expression were downregulated by treatment with AbBmi-1 in MG-63 osteosarcoma cells. In conclusion, the data provides evidence that AbBmi-1 inhibited the progression of osteosarcoma, suggesting that AbBmi-1 may be a novel anti-cancer agent through the inhibition of Bmi-1 via activating the NF-κB pathway in osteosarcoma. PMID:28983587

  1. Sodium phenylbutyrate ameliorates focal cerebral ischemic/reperfusion injury associated with comorbid type 2 diabetes by reducing endoplasmic reticulum stress and DNA fragmentation.

    Science.gov (United States)

    Srinivasan, Krishnamoorthy; Sharma, Shyam S

    2011-11-20

    Endoplasmic reticulum (ER) stress has been postulated to play a crucial role in the pathophysiology of cerebral ischemic/reperfusion (I/R) injury and diabetes. Diabetes is a major risk factor and also common amongst the people who suffer from stroke. In this study, we have investigated the neuroprotective potential of sodium 4-phenylbutyrate (SPB; 30-300mg/kg), a chemical chaperone by targeting ER stress in a rat model of transient focal cerebral ischemia associated with comorbid type 2 diabetes. Intraperitoneal treatment with SPB (100 and 300mg/kg) significantly ameliorated brain I/R damage as evidenced by reduction in cerebral infarct and edema volume. It also significantly improved the functional recovery of various neurobehavioral impairments (neurological deficit score, grip strength and rota rod) evoked by I/R compared with vehicle-treatment. Further, SPB (100mg/kg) significantly reduced the DNA fragmentation as shown by prominent reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells. This effect was observed concomitantly with significant attenuation in upregulation of 78kDa glucose regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153) and activation of caspase-12, specific markers of ER stress/apoptosis. The neuroprotection observed with SPB was independent of its effect on cerebral blood flow and blood glucose. In conclusion, this study demonstrates the neuroprotective effect of SPB owing to amelioration of ER stress and DNA fragmentation. It also suggest that targeting ER stress might offer a promising therapeutic approach and benefits against ischemic stroke associated with comorbid type 2 diabetes. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. A novel model for rapid induction of apoptosis in spiral ganglions of mice.

    Science.gov (United States)

    Lee, Ji Eun; Nakagawa, Takayuki; Kim, Tae Soo; Iguchi, Fukuichiro; Endo, Tsuyoshi; Dong, Youyi; Yuki, Kazuo; Naito, Yasushi; Lee, Sang Heun; Ito, Juichi

    2003-06-01

    The survival of the spiral ganglion (SG) is a critical issue in preservation of hearing. Research on topics related to this issue requires a mouse experimental model because such a model has advantages including use of genetic information and knockout or "knockin" mice. Thus, the aim of the study was to establish a mouse model for induction of apoptosis of SG neurons with a definite time course. Laboratory study using experimental animals. C57BL/6 mice were used as experimental animals and were subjected to direct application of cisplatin into the inner ear. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunostaining for Neurofilament 200-kD (NF) and peripherin were used for analysis of SG degeneration. In addition, generation of peroxynitrite in affected spiral ganglions was examined by immunostaining for nitrotyrosine. Cellular location of activated caspase-9 and cytochrome-c in dying SG neurons were examined for analysis of cell death pathway. The TUNEL assay and immunohistochemical analysis for NF and peripherin indicated that type I neurons in spiral ganglions were deleted through the apoptotic pathway over time. Spiral ganglion neurons treated with cisplatin exhibited expression of nitrotyrosine, indicating induction of peroxynitrite by cisplatin. In dying SG neurons, expression of activated caspase-9 and translocation of cytochrome-c from mitochondria to cytoplasm were observed, indicating the mitochondrial pathway of apoptosis. The predictable fashion of induction of apoptosis in SG neurons over a well-defined time course in the model in the study will aid studies of the molecular mechanism of cell death and elucidation of a strategy for prevention of SG degeneration.

  3. The leaves of Diospyros kaki exert beneficial effects on a benzalkonium chloride-induced murine dry eye model.

    Science.gov (United States)

    Kim, Kyung-A; Hyun, Lee Chung; Jung, Sang Hoon; Yang, Sung Jae

    2016-01-01

    In this study, the beneficial effects of the oral administration of ethanol extract of Diospyros kaki (EEDK) were tested on a mouse dry eye model induced by benzalkonium chloride (BAC). A solution of 0.2% BAC was administered topically to mouse eyes for 14 days, twice daily, to induce dry eye. Various concentrations of EEDK were administrated daily by oral gavage for 14 days after BAC treatment. Preservative-free eye drops were instilled in the positive-control group. The tear secretion volume (Schirmer's test), tear break-up time (BUT), and fluorescein score were measured on the ocular surface. BAC-induced corneal damage was tested with hematoxylin-eosin staining. Moreover, apoptotic cell death in the corneal epithelial layer was investigated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The protein expression level of interleukin-1alpha (IL-1α), IL-1β, IL-6, tumor necrosis factor- alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) was determined with western blot analysis. Furthermore, squamous metaplasia in the corneal epithelial layer was detected with immunofluorescent staining for cytokeratine-10. The cellular proliferation in the cornea was examined with immunohistochemical staining for Ki-67. EEDK treatment resulted in prolonged BUT, decreased fluorescein score, increased tear volume, and smoother epithelial cells compared with BAC treatment alone in the cornea. Moreover, EEDK treatment inhibited the inflammatory response and corneal epithelial cell death in a BAC-induced murine dry eye model, and changes in squamous cells were inhibited. Proliferative activity in the corneal epithelium cells was improved with EEDK. EEDK could be a potential therapeutic agent in the clinical treatment of dry eye.

  4. Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

    International Nuclear Information System (INIS)

    Chen Wenshu; Lee Yijang; Yu Yichu; Hsaio Chinghui

    2010-01-01

    Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of γ-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G 2 /M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

  5. Common variants in mismatch repair genes associated with increased risk of sperm DNA damage and male infertility

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    Ji Guixiang

    2012-05-01

    Full Text Available Abstract Background The mismatch repair (MMR pathway plays an important role in the maintenance of the genome integrity, meiotic recombination and gametogenesis. This study investigated whether genetic variations in MMR genes are associated with an increased risk of sperm DNA damage and male infertility. Methods We selected and genotyped 21 tagging single nucleotide polymorphisms (SNPs in five MMR genes (MLH1, MLH3, PMS2, MSH4 and MSH5 using the SNPstream 12-plex platform in a case-control study of 1,292 idiopathic infertility patients and 480 fertile controls in a Chinese population. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL assay in 450 cases. Fluorescence resonance energy transfer (FRET and co-immunoprecipitation techniques were employed to determine the effects of functional variants. Results One intronic SNP in MLH1 (rs4647269 and two non-synonymous SNPs in PMS2 (rs1059060, Ser775Asn and MSH5 (rs2075789, Pro29Ser seem to be risk factors for the development of azoospermia or oligozoospermia. Meanwhile, we also identified a possible contribution of PMS2 rs1059060 to the risk of male infertility with normal sperm count. Among patients with normal sperm count, MLH1 rs4647269 and PMS2 rs1059060 were associated with increased sperm DNA damage. Functional analysis revealed that the PMS2 rs1059060 can affect the interactions between MLH1 and PMS2. Conclusions Our results provide evidence supporting the involvement of genetic polymorphisms in MMR genes in the aetiology of male infertility.

  6. Assessment of the proliferative, apoptotic and cellular renovation indices of the human mammary epithelium during the follicular and luteal phases of the menstrual cycle

    International Nuclear Information System (INIS)

    Navarrete, Maria Alicia H; Maier, Carolina M; Falzoni, Roberto; Quadros, Luiz Gerk de Azevedo; Lima, Geraldo R; Baracat, Edmund C; Nazário, Afonso CP

    2005-01-01

    During the menstrual cycle, the mammary gland goes through sequential waves of proliferation and apoptosis. In mammary epithelial cells, hormonal and non-hormonal factors regulate apoptosis. To determine the cyclical effects of gonadal steroids on breast homeostasis, we evaluated the apoptotic index (AI) determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in human mammary epithelial cells during the spontaneous menstrual cycle and correlated it with cellular proliferation as determined by the expression of Ki-67 during the same period. Normal breast tissue samples were obtained from 42 randomly selected patients in the proliferative (n = 21) and luteal (n = 21) phases. Menstrual cycle phase characterization was based on the date of the last and subsequent menses, and on progesterone serum levels obtained at the time of biopsy. The proliferation index (PI), defined as the number of Ki-67-positive nuclei per 1,000 epithelial cells, was significantly larger in the luteal phase (30.46) than in the follicular phase (13.45; P = 0.0033). The AI was defined as the number of TUNEL-positive cells per 1,000 epithelial cells. The average AI values in both phases of the menstrual cycle were not statistically significant (P = 0.21). However, the cell renewal index (CRI = PI/AI) was significantly higher in the luteal phase (P = 0.033). A significant cyclical variation of PI, AI and CRI was observed. PI and AI peaks occurred on about the 24th day of the menstrual cycle, whereas the CRI reached higher values on the 28th day. We conclude that proliferative activity is dependent mainly on hormonal fluctuations, whereas apoptotic activity is probably regulated by hormonal and non-hormonal factors

  7. Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

    Directory of Open Access Journals (Sweden)

    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. A randomized, placebo-controlled, preoperative trial of allopurinol in subjects with colorectal adenoma.

    Science.gov (United States)

    Puntoni, Matteo; Branchi, Daniela; Argusti, Alessandra; Zanardi, Silvia; Crosta, Cristiano; Meroni, Emanuele; Munizzi, Francesco; Michetti, Paolo; Coccia, Gianni; De Roberto, Giuseppe; Bandelloni, Roberto; Turbino, Laura; Minetti, Egle; Mori, Marco; Salvi, Sandra; Boccardo, Simona; Gatteschi, Beatrice; Benelli, Roberto; Sonzogni, Angelica; DeCensi, Andrea

    2013-02-01

    Inflammation and oxidative stress play a crucial role in the development of colorectal cancer (CRC) and interference with these mechanisms represents a strategy in CRC chemoprevention. Allopurinol, a safe molecular scavenger largely used as antigout agent, has been shown to increase survival of patients with advanced CRC and to reduce CRC incidence in long-term gout users in epidemiologic studies. We conducted a randomized, double-blind, placebo-controlled preoperative trial in subjects with colorectal adenomatous polyps to assess the activity of allopurinol on biomarkers of colorectal carcinogenesis. After complete colonoscopy and biopsy of the index polyp, 73 subjects with colorectal adenomas were assigned to either placebo or one of two doses of allopurinol (100 mg or 300 mg) and treated for four weeks before polyp removal. Change of Ki-67 labeling index in adenomatous tissue was the primary endpoint. Secondary endpoints were the immunohistochemical (IHC) expression of NF-κB, β-catenin, topoisomerase-II-α, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in adenomatous polyps and normal adjacent colonic tissue. Compared with placebo, Ki-67 levels were not significantly modulated by allopurinol, whereas β-catenin and NF-κB expression levels decreased significantly in adenomatous tissue, with a mean change from baseline of -10.6%, 95% confidence interval (CI), -20.5 to -0.7, and -8.1%, 95% CI, -22.7 to 6.5, respectively. NF-κB also decreased significantly in normal adjacent tissue (-16.4%; 95% CI, -29.0 to -3.8). No dose-response relationship was noted, except for NF-κB expression in normal tissue. Allopurinol can inhibit biomarkers of oxidative activation in colon adenomatous polyps and normal adjacent tissue. Further studies should define its potential chemopreventive activity.

  9. Administration of Tauroursodeoxycholic Acid Attenuates Early Brain Injury via Akt Pathway Activation

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    Dongdong Sun

    2017-07-01

    Full Text Available Traumatic brain injury (TBI is one of the leading causes of trauma-induced mortality and disability, and emerging studies have shown that endoplasmic reticulum (ER stress plays an important role in the pathophysiology of TBI. Tauroursodeoxycholic acid (TUDCA, a hydrophilic bile acid, has been reported to act as an ER stress inhibitor and chemical chaperone and to have the potential to attenuate apoptosis and inflammation. To study the effects of TUDCA on brain injury, we subjected mice to TBI with a controlled cortical impact (CCI device. Using western blotting, we first examined TBI-induced changes in the expression levels of GRP78, an ER stress marker, p-PERK, PERK, p-eIF2a, eIF2a, ATF4, p-Akt, Akt, Pten, Bax, Bcl-2, Caspase-12 and CHOP, as well as changes in the mRNA levels of Akt, GRP78, Caspase-12 and CHOP using RT-PCR. Neuronal cell death was assessed by a terminal deoxynucleotidyl transferase (TdT-mediated dUTP nick end-labeling (TUNEL assay, and CHOP expression in neuronal cells was detected by double-immunofluorescence staining. Neurological and motor deficits were assessed by modified neurological severity scores (mNSS and beam balance and beam walking tests, and brain water content was also assessed. Our results indicated that ER stress peaked at 72 h after TBI and that TUDCA abolished ER stress and inhibited p-PERK, p-eIF2a, ATF4, Pten, Caspase-12 and CHOP expression levels. Moreover, our results show that TUDCA also improved neurological function and alleviated brain oedema. Additionally, TUDCA increased p-Akt expression and the Bcl-2/Bax ratio. However, the administration of the Akt inhibitor MK2206 or siRNA targeting of Akt abolished the beneficial effects of TUDCA. Taken together, our results indicate that TUDCA may attenuate early brain injury via Akt pathway activation.

  10. Inhibition of HDAC6 protects against rhabdomyolysis-induced acute kidney injury.

    Science.gov (United States)

    Shi, Yingfeng; Xu, Liuqing; Tang, Jinhua; Fang, Lu; Ma, Shuchen; Ma, Xiaoyan; Nie, Jing; Pi, Xiaoling; Qiu, Andong; Zhuang, Shougang; Liu, Na

    2017-03-01

    Histone deacetylase 6 (HDAC6) inhibition has been reported to protect against ischemic stroke and prolong survival after sepsis in animal models. However, it remains unknown whether HDAC6 inhibition offers a renoprotective effect after acute kidney injury (AKI). In this study, we examined the effect of tubastatin A (TA), a highly selective inhibitor of HDAC6, on AKI in a murine model of glycerol (GL) injection-induced rhabdomyolysis. Following GL injection, the mice developed severe acute tubular injury as indicated by renal dysfunction; expression of neutrophil gelatinase-associated lipocalin (NGAL), an injury marker of renal tubules; and an increase of TdT-mediated dUTP nick-end labeling (TUNEL)-positive tubular cells. These changes were companied by increased HDAC6 expression in the cytoplasm of renal tubular cells. Administration of TA significantly reduced serum creatinine and blood urea nitrogen levels as well as attenuated renal tubular damage in injured kidneys. HDAC6 inhibition also resulted in decreased expression of NGAL, reduced apoptotic cell, and inactivated caspase-3 in the kidney after acute injury. Moreover, injury to the kidney increased phosphorylation of nuclear factor (NF)-κB and expression of multiple cytokines/chemokines including tumor necrotic factor-α and interleukin-6 and monocyte chemoattractant protein-1, as well as macrophage infiltration. Treatment with TA attenuated all those responses. Finally, HDAC6 inhibition reduced the level of oxidative stress by suppressing malondialdehyde (MDA) and preserving expression of superoxide dismutase (SOD) in the injured kidney. Collectively, these data indicate that HDAC6 contributes to the pathogenesis of rhabdomyolysis-induced AKI and suggest that HDAC6 inhibitors have therapeutic potential for AKI treatment. Copyright © 2017 the American Physiological Society.

  11. Intestinal epithelial apoptosis initiates gut mucosal injury during extracorporeal membrane oxygenation in the newborn piglet.

    Science.gov (United States)

    MohanKumar, Krishnan; Killingsworth, Cheryl R; McIlwain, R Britt; Timpa, Joseph G; Jagadeeswaran, Ramasamy; Namachivayam, Kopperuncholan; Kurundkar, Ashish R; Kelly, David R; Garzon, Steven A; Maheshwari, Akhil

    2014-02-01

    Neonates and young infants exposed to extracorporeal circulation during extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass are at risk of developing a systemic inflammatory response syndrome with multi-organ dysfunction. We used a piglet model of ECMO to investigate the hypothesis that epithelial apoptosis is an early event that precedes villous damage during ECMO-related bowel injury. Healthy 3-week-old piglets were subjected to ECMO for up to 8 h. Epithelial apoptosis was measured in histopathological analysis, nuclear imaging, and terminal deoxynucleotidyl transferase dUTP nick end labeling. Plasma intestinal fatty acid-binding protein (I-FABP) levels were measured by enzyme immunoassay. Intestinal mast cells were isolated by fluorescence-assisted cell sorting. Cleaved caspase-8, caspase-9, phospho-p38 MAPK, and fas ligand expression were investigated by immunohistochemistry, western blots, and reverse transcriptase-quantitative PCR. Piglet ECMO was associated with increased gut epithelial apoptosis. Extensive apoptotic changes were noted on villus tips and in scattered crypt cells after 2 h of ECMO. After 8 h, the villi were denuded and apoptotic changes were evident in a majority of crypt cells. Increased circulating I-FABP levels, a marker of gut epithelial injury, showed that epithelial injury occurred during ECMO. We detected increased cleaved caspase-8, but not cleaved caspase-9, in epithelial cells indicating that the extrinsic apoptotic pathway was active. ECMO was associated with increased fas ligand expression in intestinal mast cells, which was induced through activation of the p38 mitogen-activated protein kinase. We conclude that epithelial apoptosis is an early event that initiates gut mucosal injury in a piglet model of ECMO.

  12. Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons

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    Chintala, Shravan; Cheng, Mei; Zhang, Xiao

    2015-01-01

    The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and

  13. Increased endothelial apoptotic cell density in human diabetic erectile tissue--comparison with clinical data.

    Science.gov (United States)

    Costa, Carla; Soares, Raquel; Castela, Angela; Adães, Sara; Hastert, Véronique; Vendeira, Pedro; Virag, Ronald

    2009-03-01

    Erectile dysfunction (ED) is a common complication of diabetes. Endothelial cell (EC) dysfunction is one of the main mechanisms of diabetic ED. However, loss of EC integrity has never been assessed in human diabetic corpus cavernosum. To identify and quantify apoptotic cells in human diabetic and normal erectile tissue and to compare these results with each patient's clinical data and erection status. Eighteen cavernosal samples were collected, 13 from diabetics with ED and 5 from nondiabetic individuals. Cavernosal structure and cell proliferation status were evaluated by immunohistochemistry. Tissue integrity was assessed by terminal transferase dUTP nick end labeling assay, an index of apoptotic cell density (ACD) established and compared with each patient age, type of diabetes, arterial risk factors number, arterial/veno-occlusive disease, response to intracavernous vasoactive injections (ICI), and penile nitric oxide release test (PNORT). Establish an index of ACD and correlate those results with patient clinical data. Nondiabetic samples presented few scattered cells in apoptosis and an ACD of 7.15 +/- 0.44 (mean apoptotic cells/tissue area mm(2) +/- standard error). The diabetic group showed an increased ACD of 23.82 +/- 1.53, and apoptotic cells were located specifically at vascular sites. Rehabilitation of these endothelial lesions seemed impaired, as no evidence of EC proliferation was observed. Furthermore, higher ACD in diabetic individuals correlated to poor response to PNORT and to ICI. We provided evidence for the first time that loss of cavernosal EC integrity is a crucial event involved in diabetic ED. Furthermore, we were able to establish a threshold between ACD values and cavernosal tissue functionality, as assessed by PNORT and vasoactive ICI.

  14. Cyclosporine a inhibits apoptosis of rat gingival epithelium.

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    Ma, Su; Liu, Peihong; Li, Yanwu; Hou, Lin; Chen, Li; Qin, Chunlin

    2014-08-01

    The use of cyclosporine A (CsA) induces hyperplasia of the gingival epithelium in a site-specific response manner, but the molecular mechanism via which the lesion occurs is unclear. The present research aims to investigate the site-specific effect of CsA on the apoptosis of gingival epithelium associated with gingival hyperplasia. Forty Wistar rats were divided into CsA-treated and non-treated groups. Paraffin-embedded sections of mandibular first molars were selected for hematoxylin and eosin staining, immunohistochemistry analyses of bcl-2 and caspase-3, and the staining of terminal deoxynucleotidyl transfer-mediated dUTP nick-end labeling (TUNEL). The area of the whole gingival epithelium and the length of rete pegs were measured, and the number of bcl-2- and caspase-3-positive cells in the longest rete peg were counted. The analysis of variance for factorial designs and Fisher least significant difference test for post hoc analysis were used to determine the significance levels. In CsA-treated rats, bcl-2 expression was significantly upregulated, whereas caspase-3 expression was downregulated, along with a reduced number of TUNEL-positive cells. The site-specific distribution of bcl-2 was consistent with the site-specific hyperplasia of the gingival epithelium in CsA-treated rats. CsA inhibited gingival epithelial apoptosis via the mitochondrial pathway and common pathway. The antiapoptotic protein bcl-2 might play a critical role in the pathogenesis of the site-specific hyperplasia of gingival epithelium induced by CsA. There were mechanistic differences in the regulation of apoptosis for cells in the attached gingival epithelium, free gingival epithelium, and junctional epithelium.

  15. [Inhibitory effect of baicalein on the proliferation and invasion of osteosarcoma cells and mechanism].

    Science.gov (United States)

    Tang, Zhibin; Li, Chun; Chen, Zhiwei

    2015-03-01

    To explore the effect of baicalein on the proliferation and invasion of osteosarcoma cells and its related mechanism. Osteosarcoma MG-63 cells that were cultured in vitro were respectively treated with 20 μL culture medium (control group), dehydrated alcohol (0 μmol/L baicalein group), 100 and 200 μmol/L baicalein solution for 48 hours. Cell proliferation was analyzed by MTT assay. The cell invasion ability was detected using Transwell(TM) invasion assay. The expression of ezrin mRNA was examined by real-time quantitative PCR. The expressions of ezrin protein and p-ezrin protein were measured using Western blotting. Apoptosis index (AI) was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The inhibitory rates of cell proliferation significantly increased in 100 and 200 μmol/L baicalein groups as compared with 0 μmol/L baicalein group. Moreover, that was higher in 200 μmol/L baicalein group than in 100 μmol/L baicalein group. In comparison with control and 0 μmol/L baicalein groups, the mean cell numbers of permeated membrane and levels of ezrin mRNA, ezrin protein and p-ezrin protein gradually decreased, but AI was gradually elevated with the increase of baicalein concentrations, whereas there was no significant difference in these indicators between 0 μmol/L baicalein group and control group. Baicalein can inhibit the proliferation and invasion of osteosarcoma MG-63 cells. The mechanism may be associated with the inhibited expression and activity of ezrin protein and the promoted tumor cell apoptosis.

  16. An experimental study on the antitumor effect of 131I-17-AAG in vitro and in vivo.

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    Wenyong, Tu; Lu, Liu; Daozhen, Chen; Weidong, Yin; Ying, Huang

    2009-02-01

    To observe the antitumor effect of (131)I-17-allylamino-17-demethoxygeldanamycin ((131)I-17-AAG) in vitro/in vivo and explore its antitumor mechanism with a view to its potential therapeutic application. (131)I-17-AAG was prepared by the reaction of 17-AAG with Na [(131)I] in the presence of hydrogen peroxide. The effects of (131)17-AAG on cell growth inhibition and cell cycle distribution in vitro were studied in BEL-7402 cells lines. Following BEL-7402 tumor implantation by subcutaneous xenografts into nude mice, the reagents were injected through the tail vein, and the tumor volume was measured and analyzed. At the end of the experiment, tumor specimens were processed for histopathological analysis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis. The expression change of Akt2 was tested by Western-blot analysis. Methyl-thiazolyl-tetrazolium assay showed inhibition rates of 27.7 +/- 5.3%, 57.3 +/- 4.3%, and 63.7 +/- 3.1%, in Na(131)I group, 17-AAG group, and (131)I-17-AAG group, respectively. The inhibition rate in the (131)I-17-AAG group differed significantly between N(a131)I group and 17-AAG group (F = 229.49, P AAG group, and (131)I-17-AAG group, respectively. Following infusion for 32 days, the tumor volumes in the (131)I-17-AAG group were significantly smaller than those in the DMSO group (F = 24.18, P AAG inhibited the proliferation of tumor cells and induced apoptosis. The expression of Akt2 in (131)I-17-AAG was significantly lower than that in the DMSO group or (131)I group. (131)I-17-AAG can effectively inhibit the growth of BEL-7402 tumor cells in vitro and in vivo. (131)I-17-AAG is a promising agent for the treatment of BEL-7402 cell tumor.

  17. Bisphenol A induces spermatocyte apoptosis in rare minnow Gobiocypris rarus

    International Nuclear Information System (INIS)

    Zhang, Yingying; Cheng, Mengqian; Wu, Lang; Zhang, Guo; Wang, Zaizhao

    2016-01-01

    Highlights: • Adult male G. rarus were exposed to 225 μg/L BPA for 7, 21 and 63 days. • BPA could induce spermatocyte apoptosis in rare minnow testis. • The mitochondrial apoptotic pathway participated in the germ cell apoptosis. • The spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest. - Abstract: Bisphenol A (BPA) is an endocrine disruptor, and could induce germ cells apoptosis in the testis of mammals. But whether it could affect fish in the same mechanism has not’ been studied till now. In the present study, to investigate the influence of BPA on testis germ cells in fish, adult male rare minnow Gobiocypris rarus were exposed to 225 μg L"−"1 (0.99 μM) BPA for 1, 3 and 9 weeks. Through TdT-mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM) analysis, we found that the amount of apoptotic spermatocytes significantly increased in a time dependent manner following BPA exposure. Western Blot results showed that the ratio of Bcl2/Bax, the important apoptosis regulators in intrinsic mitochondrial apoptotic pathway, was significantly decreased. qPCR showed that mRNA expression of several genes in mitochondrial apoptotic pathway including bcl2, bax, casp9, cytc and mcl1b were significantly changed following BPA exposure. In addition, mRNA expression of meiosis regulation genes (kpna7 and wee2), and genes involved in both apoptosis and meiosis (birc5, ccna1, and gsa1a) were also affected by BPA. Taken together, the present study demonstrated that BPA could induce spermatocytes apoptosis in rare minnow testis, and the apoptosis was probably under regulation of intrinsic mitochondrial apoptotic pathway. Moreover, the spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest.

  18. Paricalcitol attenuates lipopolysaccharide-induced inflammation and apoptosis in proximal tubular cells through the prostaglandin E₂ receptor EP4

    Directory of Open Access Journals (Sweden)

    Yu Ah Hong

    2017-06-01

    Full Text Available Background: Vitamin D is considered to exert a protective effect on various renal diseases but its underlying molecular mechanism remains poorly understood. This study aimed to determine whether paricalcitol attenuates inflammation and apoptosis during lipopolysaccharide (LPS-induced renal proximal tubular cell injury through the prostaglandin E₂ (PGE₂ receptor EP4. Methods: Human renal tubular epithelial (HK-2 cells were pretreated with paricalcitol (2 ng/mL for 1 hour and exposed to LPS (1 μg/mL. The effects of paricalcitol pretreatment in relation to an EP4 blockade using AH-23848 or EP4 small interfering RNA (siRNA were investigated. Results: The expression of cyclooxygenase-2, PGE₂, and EP4 were significantly increased in LPS-exposed HK-2 cells treated with paricalcitol compared with cells exposed to LPS only. Paricalcitol prevented cell death induced by LPS exposure, and the cotreatment of AH-23848 or EP4 siRNA offset these cell-protective effects. The phosphorylation and nuclear translocation of p65 nuclear factor-kappaB (NF-κB were decreased and the phosphorylation of Akt was increased in LPS-exposed cells with paricalcitol treatment. AH-23848 or EP4 siRNA inhibited the suppressive effects of paricalcitol on p65 NF-κB nuclear translocation and the activation of Akt. The production of proinflammatory cytokines and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were attenuated by paricalcitol in LPS exposed HK-2 cells. The cotreatment with an EP4 antagonist abolished these anti-inflammatory and antiapoptotic effects. Conclusion: EP4 plays a pivotal role in anti-inflammatory and antiapoptotic effects through Akt and NF-κB signaling after paricalcitol pretreatment in LPS-induced renal proximal tubule cell injury.

  19. Influence of mild traumatic brain injury during pediatric stage on short-term memory and hippocampal apoptosis in adult rats.

    Science.gov (United States)

    Park, Mi-Sook; Oh, Hyean-Ae; Ko, Il-Gyu; Kim, Sung-Eun; Kim, Sang-Hoon; Kim, Chang-Ju; Kim, Hyun-Bae; Kim, Hong

    2014-06-01

    Traumatic brain injury (TBI) is a leading cause of neurological deficit in the brain, which induces short- and long-term brain damage, cognitive impairment with/without structural alteration, motor deficits, emotional problems, and death both in children and adults. In the present study, we evaluated whether mild TBI in childhood causes persisting memory impairment until adulthood. Moreover, we investigated the influence of mild TBI on memory impairment in relation with hippocampal apoptosis. For this, step-down avoidance task, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry for caspase-3 were performed. Male Sprague-Dawley rats were used in the experiments. The animals were randomly divided into two groups: sham-operation group and TBI-induction group. The mild TBI model was created with an electromagnetic contusion device activated at a velocity of 3.0 m/sec. The results showed that mild TBI during the pediatric stage significantly decreased memory retention. The numbers of TUNEL-positive and caspase-3-positive cells were increased in the TBI-induction group compared to those in the sham-operation group. Defective memory retention and apoptosis sustained up to the adult stage. The present results shows that mild TBI induces long-lasting cognitive impairment from pediatric to adult stages in rats through the high level of apoptosis. The finding of this study suggests that children with mild TBI may need intensive treatments for the reduction of long-lasting cognitive impairment by secondary neuronal damage.

  20. Effect of dose rate on residual γ-H2AX levels and frequency of micronuclei in X-irradiated mouse lymphocytes.

    Science.gov (United States)

    Turner, H C; Shuryak, I; Taveras, M; Bertucci, A; Perrier, J R; Chen, C; Elliston, C D; Johnson, G W; Smilenov, L B; Amundson, S A; Brenner, D J

    2015-03-01

    The biological risks associated with low-dose-rate (LDR) radiation exposures are not yet well defined. To assess the risk related to DNA damage, we compared the yields of two established biodosimetry end points, γ-H2AX and micronuclei (MNi), in peripheral mouse blood lymphocytes after prolonged in vivo exposure to LDR X rays (0.31 cGy/min) vs. acute high-dose-rate (HDR) exposure (1.03 Gy/min). C57BL/6 mice were total-body irradiated with 320 kVP X rays with doses of 0, 1.1, 2.2 and 4.45 Gy. Residual levels of total γ-H2AX fluorescence in lymphocytes isolated 24 h after the start of irradiation were assessed using indirect immunofluorescence methods. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptotic cell frequency in lymphocytes sampled at 24 h. Curve fitting analysis suggested that the dose response for γ-H2AX yields after acute exposures could be described by a linear dependence. In contrast, a linear-quadratic dose-response shape was more appropriate for LDR exposure (perhaps reflecting differences in repair time after different LDR doses). Dose-rate sparing effects (P effect across the dose range 24 h or 7 days post exposure. In conclusion, the γ-H2AX biomarker showed higher sensitivity to measure dose-rate effects after low-dose LDR X rays compared to MNi formation; however, confounding factors such as variable repair times post exposure, increased cell killing and cell cycle block likely contributed to the yields of MNi with accumulating doses of ionizing radiation.

  1. Effects of hepatitis B virus S protein exposure on sperm membrane integrity and functions.

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    XiangJin Kang

    Full Text Available BACKGROUND: Hepatitis B is a public health problem worldwide. Viral infection can affect a man's fertility, but only scant information about the influence of hepatitis B virus (HBV infection on sperm quality is available. The purpose of this study was to investigate the effect of hepatitis B virus S protein (HBs on human sperm membrane integrity and functions. METHODS/PRINCIPAL FINDINGS: Reactive oxygen species (ROS, lipid peroxidation (LP, total antioxidant capacity (TAC and phosphatidylserine (PS externalization were determined. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assays and flow cytometric analyses were performed. (1 After 3 h incubation with 25 µg/ml of HBs, the average rates of ROS positive cells, annexin V-positive/propidium iodide (PI-negative cells, Caspases-3,-8,-9 positive cells and TUNEL-positive cells were significantly increased in the test groups as compared to those in the control groups, while TAC level was decreased when compared with the control. The level of malondialdehyde (MDA in the sperm cells exposed to 50 µg/ml of HBs for 3 h was significantly higher than that in the control (P<0.05-0.01. (2 HBs increased the MDA levels and the numbers of ROS positive cells, annexin V-positive/PI-negative cells, caspases-3, -8, -9 positive cells and TUNEL-positive cells in a dose-dependent manner. (3 HBs monoclonal antibody (MAb and N-Acetylcysteine (NAC reduced the number of ROS-positive sperm cells. (4 HBs decreased the TAC levels in sperm cells in a dose-dependent manner. CONCLUSION: HBs exposure could lead to ROS generation, lipid peroxidation, TAC reduction, PS externalization, activation of caspases, and DNA fragmentation, resulting in increased apoptosis of sperm cells and loss of sperm membrane integrity and causing sperm dysfunctions.

  2. Curcumin ameliorates cardiac dysfunction induced by mechanical trauma.

    Science.gov (United States)

    Li, Xintao; Cao, Tingting; Ma, Shuo; Jing, Zehao; Bi, Yue; Zhou, Jicheng; Chen, Chong; Yu, Deqin; Zhu, Liang; Li, Shuzhuang

    2017-11-05

    Curcumin, a phytochemical component derived from turmeric (Carcuma longa), has been extensively investigated because of its anti-inflammatory and anti-oxidative properties. Inflammation and oxidative stress play critical roles in posttraumatic cardiomyocyte apoptosis, which contributes to secondary cardiac dysfunction. This research was designed to identify the protective effect of curcumin on posttraumatic cardiac dysfunction and investigate its underlying mechanism. Noble-Collip drum was used to prepare a mechanical trauma (MT) model of rats, and the hemodynamic responses of traumatized rats were observed by ventricular intubation 12h after trauma. Myocardial apoptosis was determined through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay. Tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS) generated by monocytes and myocardial cells were identified through enzyme-linked immunosorbent assay (ELISA), and the intracellular alteration of Ca 2+ in cardiomyocytes was examined through confocal microscopy. In vivo, curcumin effectively ameliorated MT-induced secondary cardiac dysfunction and significantly decreased the apoptotic indices of the traumatized myocardial cells. In vitro, curcumin inhibited TNF-α production by monocytes and reduced the circulating TNF-α levels. With curcumin pretreatment, ROS production and Ca 2+ overload in H9c2 cells were attenuated when these cells were incubated with traumatic plasma. Therefore, curcumin can effectively ameliorate MT-induced cardiac dysfunction mainly by inhibiting systemic inflammatory responses and by weakening oxidative stress reaction and Ca 2+ overload in cardiomyocytes. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. 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.

  4. The radical scavenger edaravone improves neurologic function and perihematomal glucose metabolism after acute intracerebral hemorrhage.

    Science.gov (United States)

    Shang, Hanbing; Cui, Derong; Yang, Dehua; Liang, Sheng; Zhang, Weifeng; Zhao, Weiguo

    2015-01-01

    Oxidative injury caused by reactive oxygen species plays an important role in the progression of intracerebral hemorrhage (ICH)-induced secondary brain injury. Previous studies have demonstrated that the free radical scavenger edaravone may prevent neuronal injury and brain edema after ICH. However, the influence of edaravone on cerebral metabolism in the early stages after ICH and the underlying mechanism have not been fully investigated. In the present study, we investigated the effect of edaravone on perihematomal glucose metabolism using (18)F-fluorordeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT). Additionally, the neurologic deficits, brain edemas, and cell death that followed ICH were quantitatively analyzed. After blood infusion, the rats treated with edaravone showed significant improvement in both forelimb placing and corner turn tests compared with those treated with vehicle. Moreover, the brain water content of the edaravone-treated group was significantly decreased compared with that of the vehicle group on day 3 after ICH. PET/CT images of ICH rats exhibited obvious decreases in FDG standardized uptake values in perihematomal region on day 3, and the lesion-to-normal ratio of the edaravone-treated ICH rats was significantly increased compared with that of the control rats. Calculation of the brain injury volumes from the PET/CT images revealed that the volumes of the blood-induced injuries were significantly smaller in the edaravone group compared with the vehicle group. Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling assays performed 3 days after ICH revealed that the numbers of apoptotic cells in perihematomal region of edaravone-treated ICH rats were decreased relative to the vehicle group. Thus, the present study demonstrates that edaravone has scavenging properties that attenuate neurologic behavioral deficits and brain edema in the early period of ICH. Additionally, edaravone may improve

  5. Ocular Safety of Intravitreal Connective Tissue Growth Factor Neutralizing Antibody.

    Science.gov (United States)

    Motevasseli, Tahmineh; Daftarian, Narsis; Kanavi, Mozhgan Rezaei; Ahmadieh, Hamid; Bagheri, Abouzar; Hosseini, Seyed Bagher; Ansari, Shabnam; Soheili, Zahra-Soheila

    2017-08-01

    To detect the safety of intravitreal injection of anti-connective tissue growth factor (CTGF) (IVAC) in rat eyes in order to apply this neutralizing antibody for experimental animal studies. Forty-five Lister Hooded male pigmented rats were divided into five groups that received IVAC (2 μl) corresponding to the doses of 10 (B), 20 (C), 50 (D), and 100 μg/ml (E), equal to 1.25, 2.5, 6.25, and 12.5 µg/ml of antibody concentration in rat vitreous, respectively. The sham group (A) received 2 μl of normal saline. Full field electroretinography (ERG) was performed at baseline and on days 7 and 28 after IVAC. The animals were euthanized and the corresponding eyes were subjected to routine histopathology, immunohistochemistry for glial fibrillary acidic protein (GFAP), and terminal transferase dUTP nick end-labeling (TUNEL) assay. Scotopic rod b-wave amplitude and maximal combined b-wave amplitude were 111.89 ± 71.2 and 178.57 ± 55.58 μV, respectively, at baseline which significantly reduced to 79.31 ± 52.59 and 128.73 ± 41.61 μV, respectively, after 28 days in group E (p < 0.05). There was no significant reduction of amplitudes in other groups with lower doses of anti-CTGF antibody. Retinal ganglion cells were significantly decreased in group E as compared to other groups. GFAP immune reactivity was not significant in any of the groups. TUNEL test showed inner retinal neural cell apoptosis only in group E. ERG, histopathologic, and apoptotic assays revealed no toxic effects of 10-50 μg/ml of IVAC in rat eyes. Using 100 μg/ml IVAC led to a significant toxic effect in terms of functional, histopathologic, and TUNEL findings.

  6. Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias).

    Science.gov (United States)

    McClusky, L M

    2005-01-01

    To understand the processes involved in the spatial and temporal maturation of testicular cells in Squalus acanthias, we used standard morphometry, proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) immunohistochemistry. Except for immature spermatocysts (germinal zone, GZ; early-stage pre-meiotic, E-PrM), the number of cysts in all subsequent stages and the total number of cysts in the spermatogenic progression varied seasonally. The spermatogenic cycle spans about 2 years and is interrupted by germcell clone deletion via apoptosis at the mitosis-meiosis transition in April/May, manifesting as a zone of degeneration (ZD). Rate of displacement of the ZD across the testis diameter indicates that late-stage premeiotic (L-PrM) generations 12-13 require 9-10 months to reach the mature-spermatid stage. Also, the number of cysts completing spermatogenesis is approximately 4-5-fold less than the number that entered spermatogenesis proper 2 years earlier. Pronounced gonocytogenesis in the germinal ridge was coincident with ZD formation in April/May, but it was absent in the fall when mature spermatogonial and meiotic activities had resumed. Whereas strong Sertoli cell PCNA immunoreactivity dominated the GZ cyst cell-cycle activities throughout the year, except during the spring/summer months, the spermatogonial- and Sertoli-cell PCNA indices in E-PrM cysts were inversely related. PCNA immunoreactivity in spermatocytes was seasonal and dependent on the stage of meiosis. TUNEL labelling was limited to spermatogonia and increased stage-dependently in the PrM region (L-PrM = mid-stage PrM >E-PrM >GZ), correlating with ZD formation, in a season-dependent manner. Results imply that effects of normal regulatory factors in Squalus are stage- and process-specific.

  7. Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer.

    Science.gov (United States)

    Bennett, M W; O'connell, J; O'sullivan, G C; Roche, D; Brady, C; Kelly, J; Collins, J K; Shanahan, F

    1999-02-01

    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. To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. Thirty paraffin wax embedded human gastric adenocarcinomas. 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). 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. Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.

  8. Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

    International Nuclear Information System (INIS)

    Gao, Feng-Hou; Liu, Feng; Zhao, Ying-Zheng; Fang, Yong; Chen, Fang-Yuan; Wu, Ying-Li; Hu, Xiao-Hui; Li, Wei; Liu, Hua; Zhang, Yan-Jie; Guo, Zhu-Ying; Xu, Mang-Hua; Wang, Shi-Ting; Jiang, Bin

    2010-01-01

    Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment

  9. Protective Effects of Thymoquinone against Methotrexate-Induced Germ Cell Apoptosis in Male Mice

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    Fatemeh Sheikhbahaei

    2016-12-01

    Full Text Available Background: Toxic effects of anti-cancer and other drugs on the normal tissues could be reduced by the herbal plants and their fractions. This study investigated the protective effect of thymoquinone (TQ as a fraction of Nigella sativa on methotrexate (MTX- induced germ cell apoptosis in male mice. Materials and Methods: In this experimental study, thirty male Balb/c mice were divided randomly into 5 groups (n=6. A single dose of MTX (20 mg/kg and different concentrations of TQ were administrated for 4 consecutive days. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay was performed on paraffin embedded tissue sections to analysis the occurrence of apoptosis in the testis. Reverse transcription polymerase chain reaction (RT-PCR of apoptosis-related genes was performed with RNA extracted from testes of the mice. Statistical analysis was done using one-way ANOVA. Results: In the MTX group, there was a significant increase in morphologic sign of germ cell degeneration of tubules (48 ± 0.6%, apoptotic index (AI; 2.3 ± 0.6%, as well as mRNA expression of p53 (P=0.008, caspase 8 (P=0.002, caspase 3 (P=0.005, caspase 9 (P=0.000, bax (P=0.004 and the ratio of bax/bcl-2 (P=0.000, whereas there was an decrease in the expression of bcl-2 (P=0.003, as compared to control group. In MTX+TQ groups, the data showed that different concentrations of TQ could improve the harmful effects caused by the MTX. The best protective effects were achieved in MTX+TQ (10 mg/kg. Conclusion: TQ protects testicular germ cell against MTX-induced apoptosis by affecting related genes regulation.

  10. 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

  11. Beneficial effects of ursodeoxycholic acid via inhibition of airway remodelling, apoptosis of airway epithelial cells, and Th2 immune response in murine model of chronic asthma.

    Science.gov (United States)

    Işık, S; Karaman, M; Çilaker Micili, S; Çağlayan-Sözmen, Ş; Bağrıyanık, H Alper; Arıkan-Ayyıldız, Z; Uzuner, N; Karaman, Ö

    In previous studies, anti-inflammatory, anti-apoptotic and immunomodulatory effects of ursodeoxycholic acid (UDCA) on liver diseases have been shown. In this study, we aimed to investigate the effects of UDCA on airway remodelling, epithelial apoptosis, and T Helper (Th)-2 derived cytokine levels in a murine model of chronic asthma. Twenty-seven BALB/c mice were divided into five groups; PBS-Control, OVA-Placebo, OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone. Mice in groups OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone received the UDCA (50mg/kg), UDCA (150mg/kg), and dexamethasone, respectively. Epithelium thickness, sub-epithelial smooth muscle thickness, number of mast and goblet cells of samples isolated from the lung were measured. Immunohistochemical scorings of the lung tissue for matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEG-F), transforming growth factor-beta (TGF-β), terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) and cysteine-dependent aspartate-specific proteases (caspase)-3 were determined. IL-4, IL-5, IL-13, Nitric oxide, ovalbumin-specific immunoglobulin (Ig) E levels were quantified. The dose of 150mg/kg UDCA treatment led to lower epithelial thickness, sub-epithelial smooth muscle thickness, goblet and mast cell numbers compared to placebo. Except for MMP-9 and TUNEL all immunohistochemical scores were similar in both UDCA treated groups and the placebo. All cytokine levels were significantly lower in group IV compared to the placebo. These findings suggested that the dose of 150mg/kg UDCA improved all histopathological changes of airway remodelling and its beneficial effects might be related to modulating Th-2 derived cytokines and the inhibition of apoptosis of airway epithelial cells. Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

  12. Methamphetamine-induced neurotoxicity is attenuated in transgenic mice with a null mutation for interleukin-6.

    Science.gov (United States)

    Ladenheim, B; Krasnova, I N; Deng, X; Oyler, J M; Polettini, A; Moran, T H; Huestis, M A; Cadet, J L

    2000-12-01

    Increasing evidence implicates apoptosis as a major mechanism of cell death in methamphetamine (METH) neurotoxicity. The involvement of a neuroimmune component in apoptotic cell death after injury or chemical damage suggests that cytokines may play a role in METH effects. In the present study, we examined if the absence of IL-6 in knockout (IL-6-/-) mice could provide protection against METH-induced neurotoxicity. Administration of METH resulted in a significant reduction of [(125)I]RTI-121-labeled dopamine transporters in the caudate-putamen (CPu) and cortex as well as depletion of dopamine in the CPu and frontal cortex of wild-type mice. However, these METH-induced effects were significantly attenuated in IL-6-/- animals. METH also caused a decrease in serotonin levels in the CPu and hippocampus of wild-type mice, but no reduction was observed in IL-6-/- animals. Moreover, METH induced decreases in [(125)I]RTI-55-labeled serotonin transporters in the hippocampal CA3 region and in the substantia nigra-reticulata but increases in serotonin transporters in the CPu and cingulate cortex in wild-type animals, all of which were attenuated in IL-6-/- mice. Additionally, METH caused increased gliosis in the CPu and cortices of wild-type mice as measured by [(3)H]PK-11195 binding; this gliotic response was almost completely inhibited in IL-6-/- animals. There was also significant protection against METH-induced DNA fragmentation, measured by the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeled (TUNEL) cells in the cortices. The protective effects against METH toxicity observed in the IL-6-/- mice were not caused by differences in temperature elevation or in METH accumulation in wild-type and mutant animals. Therefore, these observations support the proposition that IL-6 may play an important role in the neurotoxicity of METH.

  13. Effectiveness of sugammadex for cerebral ischemia/reperfusion injury.

    Science.gov (United States)

    Ozbilgin, Sule; Yılmaz, Osman; Ergur, Bekir Ugur; Hancı, Volkan; Ozbal, Seda; Yurtlu, Serhan; Gunenc, Sakize Ferim; Kuvaki, Bahar; Kucuk, Burcu Ataseven; Sisman, Ali Rıza

    2016-06-01

    Cerebral ischemia may cause permanent brain damage and behavioral dysfunction. The efficacy and mechanisms of pharmacological treatments administered immediately after cerebral damage are not fully known. Sugammadex is a licensed medication. As other cyclodextrins have not passed the necessary phase tests, trade preparations are not available, whereas sugammadex is frequently used in clinical anesthetic practice. Previous studies have not clearly described the effects of the cyclodextrin family on cerebral ischemia/reperfusion (I/R) damage. The aim of this study was to determine whether sugammadex had a neuroprotective effect against transient global cerebral ischemia. Animals were assigned to control, sham-operated, S 16 and S 100 groups. Transient global cerebral ischemia was induced by 10-minute occlusion of the bilateral common carotid artery, followed by 24-hour reperfusion. At the end of the experiment, neurological behavior scoring was performed on the rats, followed by evaluation of histomorphological and biochemical measurements. Sugammadex 16 mg/kg and 100 mg/kg improved neurological outcome, which was associated with reductions in both histological and neurological scores. The hippocampus TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and caspase results in the S 16 and S 100 treatment groups were significantly lower than those of the I/R group. Neurological scores in the treated groups were significantly higher than those of the I/R group. The study showed that treatment with 16 mg/kg and 100 mg/kg sugammadex had a neuroprotective effect in a transient global cerebral I/R rat model. However, 100 mg/kg sugammadex was more neuroprotective in rats. Copyright © 2016. Published by Elsevier Taiwan.

  14. Effectiveness of sugammadex for cerebral ischemia/reperfusion injury

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    Sule Ozbilgin

    2016-06-01

    Full Text Available Cerebral ischemia may cause permanent brain damage and behavioral dysfunction. The efficacy and mechanisms of pharmacological treatments administered immediately after cerebral damage are not fully known. Sugammadex is a licensed medication. As other cyclodextrins have not passed the necessary phase tests, trade preparations are not available, whereas sugammadex is frequently used in clinical anesthetic practice. Previous studies have not clearly described the effects of the cyclodextrin family on cerebral ischemia/reperfusion (I/R damage. The aim of this study was to determine whether sugammadex had a neuroprotective effect against transient global cerebral ischemia. Animals were assigned to control, sham-operated, S 16 and S 100 groups. Transient global cerebral ischemia was induced by 10-minute occlusion of the bilateral common carotid artery, followed by 24-hour reperfusion. At the end of the experiment, neurological behavior scoring was performed on the rats, followed by evaluation of histomorphological and biochemical measurements. Sugammadex 16 mg/kg and 100 mg/kg improved neurological outcome, which was associated with reductions in both histological and neurological scores. The hippocampus TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase results in the S 16 and S 100 treatment groups were significantly lower than those of the I/R group. Neurological scores in the treated groups were significantly higher than those of the I/R group. The study showed that treatment with 16 mg/kg and 100 mg/kg sugammadex had a neuroprotective effect in a transient global cerebral I/R rat model. However, 100 mg/kg sugammadex was more neuroprotective in rats.

  15. Prognostic implication of apoptosis and angiogenesis in cervical uteri cancer

    International Nuclear Information System (INIS)

    Zaghloul, Mohamed S.; El Naggar, Mervat; El Deeb, Amany; Khaled, Hussein; Mokhtar, Nadia

    2000-01-01

    Purpose: A retrospective study was performed to investigate the relationship between spontaneous apoptosis and angiogenesis uterine cervix squamous cell carcinoma patients. The prognostic value of each (and both) of these biologic parameters was also tested. Methods and Materials: The pathologic materials of 40 cervical uteri squamous cell carcinoma patients were examined and immunohistochemically stained to determine the tumor angiogenesis (tumor microvascular score), using factor VIII-related antigen, and their tumor apoptotic index (AI), using the TdT-mediated dUTP nick end-labeling (TUNEL) method. Three patients were Stage I, 18 were Stage II, 15 were Stage III, and 4 were Stage IV (FIGO classification). All patients were treated with radical radiotherapy and all had follow-up for more than 2 years. Results: The mean AI was 15.1 ± 12.8, with a median of 8.3. The mean tumor microvascular score was 3 9.7 ± 14.4, with a median of 3 8. The patients' age and tumor grade did not seem to significantly affect the prognosis. On the other hand, AI and angiogenesis (tumor microvascular score) were of high prognostic significance. The 3-year disease-free survival (DFS) rate for the patients having AI above the median was 78% (confidence interval [CI] 69-87%), compared to 32% (CI 22-42%) for those having AI below the median. The DFS was 18% (CI 9-27%) for patients having an angiogenesis score above the median, while it was 86% (CI 78-94%) for those patients having a score below the median. Conclusion: Determination of both tumor microvascular score and AI can identify patients with the best prognosis of 100% DFS (with low angiogenesis score and high AI). Women with a high score and low AI had the worst prognosis (DFS = 3%, CI 1-5%). Moreover, high AI can compensate partially for the aggressive behavior of tumors showing a high rate of angiogenesis.

  16. Acute Doxorubicin Insult in the Mouse Ovary Is Cell- and Follicle-Type Dependent

    Science.gov (United States)

    Roti Roti, Elon C.; Leisman, Scott K.; Abbott, David H.; Salih, Sana M.

    2012-01-01

    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 in the ovary

  17. Therapeutic potential of Mucuna pruriens (Linn.) on ageing induced damage in dorsal nerve of the penis and its implication on erectile function: an experimental study using albino rats.

    Science.gov (United States)

    Seppan, Prakash; Muhammed, Ibrahim; Mohanraj, Karthik Ganesh; Lakshmanan, Ganesh; Premavathy, Dinesh; Muthu, Sakthi Jothi; Wungmarong Shimray, Khayinmi; Sathyanathan, Sathya Bharathy

    2018-02-15

    To study the effect of ethanolic seed extract of Mucuna pruriens on damaged dorsal nerve of the penis (DNP) in aged rat in relation to penile erection. The rats were divided into four groups Young (3 months), Aged (24 - 28 months), Aged + M. pruriens, and Young + M. pruriens (200 mg/kg b.w/60 days) and were subjected to the hypophysial - gonadal axis, nerve conduction velocity (NCV), and penile reflex. DNP sections were stained with nitric oxide synthase (nNOS), nicotinamide adenine dinucleotide phosphate (NaDPH) diaphorase, androgen receptor (AR), and osmium tetroxide. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining, electron microscopy(EM) and histometric analyses were done. Significant disturbance in hypophysial - gonadal axis was noted in aged rat. With reduced number of myelinated fibers, diameter, vacuolization, indentation of the myelin sheath, and degeneration. nNOS and its cofactor (NaDPH diaphorase) were reduced in aged rat DNP. NCV was slow in aged rats and concomitant poor penile reflex was also noted. AR showed reduced expression in aged rat DNP when compared to young and control groups. TUNEL positive cells were increased in aged rat DNP. These pathological changes were remarkably reduced or recovered in M. pruriens treated aged rats. The results indicate a multi-factorial therapeutic activity in penile innervations towards sustaining the penile erection in the presence of the extract in aged rats and justifying the claim of traditional usage.

  18. Tamaractam, a New Bioactive Lactam from Tamarix ramosissima, Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

    Science.gov (United States)

    Yao, Yao; Jiang, Cheng-Shuai; Sun, Na; Li, Wei-Qi; Niu, Yang; Han, Huai-Qin; Miao, Zhen-Hua; Zhao, Xun-Xia; Zhao, Jing; Li, Juan

    2017-01-10

    Chemical investigation of Tamarix ramosissima Ledeb, a traditional herbal medicine used for rheumatoid arthritis (RA) treatment in northwest China, led to the discovery of a new phenolic aromatic rings substituted lactam, tamaractam ( 1 ), together with the previously reported compounds cis - N -feruloyl-3- O -methyldopamine ( 2 ) and trans - N -feruloyl-3- O -methyldopamine ( 3 ). The structures of the compounds were determined by high resolution electrospray ionization mass spectroscopy (HRESIMS) and 1D and 2D-NMR experiments, as well as comparison with the literature data. The effects of the three compounds on the viability of RA fibroblast-like synoviocytes (RA-FLS) were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2- H -tetrazolium bromide (MTT) assay. Pro-apoptosis effect of compound 1 in RA-FLS was further investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, activated caspase-3/7 level assessment using luminescence assay, and sub-G₁ fraction measurement using flow cytometry. It was found that these three compounds displayed variable proliferation inhibitory activity in RA-FLS, and compound 1 exhibited the strongest effect. Compound 1 could remarkably induce cellular apoptosis of RA-FLS, increase activated caspase-3/7 levels, and significantly increase sub-G₁ fraction in the cell cycle. The results suggested that compound 1 may inhibit the proliferation of RA-FLS through apoptosis-inducing effect, and these compounds may contribute to the anti-RA effect of T. ramosissima .

  19. Tamaractam, a New Bioactive Lactam from Tamarix ramosissima, Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

    Directory of Open Access Journals (Sweden)

    Yao Yao

    2017-01-01

    Full Text Available Chemical investigation of Tamarix ramosissima Ledeb, a traditional herbal medicine used for rheumatoid arthritis (RA treatment in northwest China, led to the discovery of a new phenolic aromatic rings substituted lactam, tamaractam (1, together with the previously reported compounds cis-N-feruloyl-3-O-methyldopamine (2 and trans-N-feruloyl-3-O-methyldopamine (3. The structures of the compounds were determined by high resolution electrospray ionization mass spectroscopy (HRESIMS and 1D and 2D-NMR experiments, as well as comparison with the literature data. The effects of the three compounds on the viability of RA fibroblast-like synoviocytes (RA-FLS were assessed by 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay. Pro-apoptosis effect of compound 1 in RA-FLS was further investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, activated caspase-3/7 level assessment using luminescence assay, and sub-G1 fraction measurement using flow cytometry. It was found that these three compounds displayed variable proliferation inhibitory activity in RA-FLS, and compound 1 exhibited the strongest effect. Compound 1 could remarkably induce cellular apoptosis of RA-FLS, increase activated caspase-3/7 levels, and significantly increase sub-G1 fraction in the cell cycle. The results suggested that compound 1 may inhibit the proliferation of RA-FLS through apoptosis-inducing effect, and these compounds may contribute to the anti-RA effect of T. ramosissima.

  20. In Vitro and In Vivo Efficacy Studies of Lavender angustifolia Essential Oil and Its Active Constituents on the Proliferation of Human Prostate Cancer

    Science.gov (United States)

    Zhao, Yunqi; Chen, Ran; Wang, Yun; Qing, Chen; Wang, Wei; Yang, Yixin

    2016-01-01

    Lavandula angustifolia is the most widely cultivated Lavandula species. The extraction of its flower and leaves has been used as herbal medicine. In this study, the in vitro antitumor activities were tested on human prostate cancer PC-3 and DU145 cell lines. Flow cytometry technology was applied to study apoptosis induction and cell cycle arrest. The PC-3 cell line was used to establish subcutaneous xenograft tumors in nude mice. Paraffin sections from xenograft tumor specimens were used in the TUNEL (terminal deocynucleotide transferase dUTP nick end labeling) assay and an immunohistochemistry assay to detect cell proliferation markers Ki67 and PCNA. Lavender essential oil, linalool, and linalyl acetate showed stronger inhibitory effect on PC-3 cells than on DU145 cells. The apoptotic cell populations observed in PC-3 cells treated with lavender essential oil, linalool, and linalyl acetate were 74.76%, 67.11%, and 56.14%, respectively. The PC-3 cells were mainly arrested in the G2/M phase. In the xenograft model with PC-3 cell transplantation, essential oil and linalool significantly suppressed tumor growth. The immunosignals of Ki67 and PCNA in the essential oil, linalool, and linalyl acetate treatment groups were significantly lower than that of the control group in xenograft tumor sections. The TUNEL assay indicated that each of the 3 phytochemicals significantly induced apoptosis compared to the control group. This study provides novel insight and evidence on the antiproliferative effect of L angustifolia essential oil and its major constituents on human prostate cancer. The antitumor effect was associated with cell proliferation inhibition and apoptosis induction in xenograft tumors. PMID:27151584

  1. Protective Effect of Unacylated Ghrelin on Compression-Induced Skeletal Muscle Injury Mediated by SIRT1-Signaling

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    Felix N. Ugwu

    2017-11-01

    Full Text Available Unacylated ghrelin, the predominant form of circulating ghrelin, protects myotubes from cell death, which is a known attribute of pressure ulcers. In this study, we investigated whether unacylated ghrelin protects skeletal muscle from pressure-induced deep tissue injury by abolishing necroptosis and apoptosis signaling and whether these effects were mediated by SIRT1 pathway. Fifteen adult Sprague Dawley rats were assigned to receive saline or unacylated ghrelin with or without EX527 (a SIRT1 inhibitor. Animals underwent two 6-h compression cycles with 100 mmHg static pressure applied over the mid-tibialis region of the right limb whereas the left uncompressed limb served as the intra-animal control. Muscle tissues underneath the compression region, and at the similar region of the opposite uncompressed limb, were collected for analysis. Unacylated ghrelin attenuated the compression-induced muscle pathohistological alterations including rounding contour of myofibers, extensive nucleus accumulation in the interstitial space, and increased interstitial space. Unacylated ghrelin abolished the increase in necroptosis proteins including RIP1 and RIP3 and attenuated the elevation of apoptotic proteins including p53, Bax, and AIF in the compressed muscle. Furthermore, unacylated ghrelin opposed the compression-induced phosphorylation and acetylation of p65 subunit of NF-kB. The anti-apoptotic effect of unacylated ghrelin was shown by a decrease in apoptotic DNA fragmentation and terminal dUTP nick-end labeling index in the compressed muscle. The protective effects of unacylated ghrelin vanished when co-treated with EX527. Our findings demonstrated that unacylated ghrelin protected skeletal muscle from compression-induced injury. The myoprotective effects of unacylated ghrelin on pressure-induced tissue injury were associated with SIRT1 signaling.

  2. Can social instability, food deprivation and food inequality accelerate neuronal aging?

    Directory of Open Access Journals (Sweden)

    Fatemeh Moradi

    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. 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 pyramidal cells to apoptosis and premature aging induced by lipofuscin accumulation. 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

  3. Identification of novel candidate compounds targeting TrkB to induce apoptosis in neuroblastoma

    International Nuclear Information System (INIS)

    Nakamura, Yohko; Suganami, Akiko; Fukuda, Mayu; Hasan, Md Kamrul; Yokochi, Tomoki; Takatori, Atsushi; Satoh, Shunpei; Hoshino, Tyuji; Tamura, Yutaka; Nakagawara, Akira

    2014-01-01

    Neuroblastoma (NB) is one of the most frequent solid tumors in children and its prognosis is still poor. The neurotrophin receptor TrkB and its ligand brain-derived neurotrophic factor (BDNF) are expressed at high levels in high-risk NBs and are involved in defining the poor prognosis of the patients. However, the TrkB targeting therapy has never been realized in the clinic. We performed an in silico screening procedure utilizing an AutoDock/grid computing technology in order to identify novel small chemical compounds targeting the BDNF-binding domain of TrkB. For the first screening, a library of three million synthetic compounds was screened in silico and was ranked according to the Docking energy. The top-ranked 37 compounds were further functionally screened for cytotoxicity by using NB cell lines. We have finally identified seven compounds that kill NB cells with the IC 50 values of 0.07–4.6 μmol/L. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that these molecules induce apoptosis accompanied by p53 activation in NB cell lines. The candidate compounds and BDNF demonstrated an antagonistic effect on cell growth, invasion, and colony formation, possibly suggesting competition at the BDNF-binding site of TrkB. The candidate compounds had tumor-suppressive activity in xenograft and in vivo toxicity tests (oral and intravenous administrations) using mice, and did not show any abnormal signs. Using in silico Docking screening we have found new candidate TrkB inhibitors against high-risk NBs, which could lead to new anti-cancer drugs

  4. EFFECT OF STENT ABSORBED c-myc ANTISENSE OLIGODEOXYNUCLEOTIDE ON SMOOTH MUSCLE CELLS APOPTOSIS IN RABBIT CAROTID ARTERY

    Institute of Scientific and Technical Information of China (English)

    张新霞; 崔长琮; 李江; 崔翰斌; 徐仓宝; 朱参战

    2002-01-01

    Objective To investigate the effect of gelatin coated Platinium-Iridium stent absorbed c-myc antisense oligodeoxynucleotide (ASODN) on smooth muscle cells apoptosis in a normal rabbit carotid arteries. Methods Gelatin coated Platinium-Iridium stents were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomly divided into control group and treated group receiving c-myc ASODN (n=16, respectively). On 7, 14, 30 and 90 days following the stenting procedure ,morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical method. Apoptotic smooth muscle cells was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Results At 7 and 14 days after stenting,there were no detectable apoptotic cells in both groups. The apoptotic cells occurred in the neointima 30 and 90 days after stenting, and the number of apoptotic cells at 30 days were less [4.50±1.29 vs 25.75±1.89 (number/0.1mm2)] than that at 90 days [13.50±1.91 vs 41.50±6.46 (number/0.1mm2)]. Meanwhile c-myc ASODN induced more apoptotic cells than the control group(P<0.0001). c-myc protein expression was weak positive or negative in treated group and positive in control group.Conclusion c-myc ASODN can induce smooth muscle cells apoptosis after stenting in normal rabbit carotid arteries,and it can be used to prevent in-stent restenosis.

  5. Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth.

    Science.gov (United States)

    Radchuk, Volodymyr; Weier, Diana; Radchuk, Ruslana; Weschke, Winfriede; Weber, Hans

    2011-01-01

    After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.

  6. Human neural progenitor cells decrease photoreceptor degeneration, normalize opsin distribution and support synapse structure in cultured porcine retina.

    Science.gov (United States)

    Mollick, Tanzina; Mohlin, Camilla; Johansson, Kjell

    2016-09-01

    Retinal neurodegenerative disorders like retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and retinal detachment decrease retinal functionality leading to visual impairment. The pathological events are characterized by photoreceptor degeneration, synaptic disassembly, remodeling of postsynaptic neurons and activation of glial cells. Despite intense research, no effective treatment has been found for these disorders. The current study explores the potential of human neural progenitor cell (hNPC) derived factors to slow the degenerative processes in adult porcine retinal explants. Retinas were cultured for 3 days with or without hNPCs as a feeder layer and investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemical, western blot and quantitative real time-polymerase chain reaction (qRT-PCR) techniques. TUNEL showed that hNPCs had the capacity to limit photoreceptor cell death. Among cone photoreceptors, hNPC coculture resulted in better maintenance of cone outer segments and reduced opsin mislocalization. Additionally, maintained synaptic structural integrity and preservation of second order calbindin positive horizontal cells was also observed. However, Müller cell gliosis only seemed to be alleviated in terms of reduced Müller cell density. Our observations indicate that at 3 days of coculture, hNPC derived factors had the capacity to protect photoreceptors, maintain synaptic integrity and support horizontal cell survival. Human neural progenitor cell applied treatment modalities may be an effective strategy to help maintain retinal functionality in neurodegenerative pathologies. Whether hNPCs can independently hinder Müller cell gliosis by utilizing higher concentrations or by combination with other pharmacological agents still needs to be determined. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Oligodendrocyte death, neuroinflammation, and the effects of minocycline in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

    Science.gov (United States)

    Mehrabian, Zara; Guo, Yan; Weinreich, Daniel; Bernstein, Steven L

    2017-01-01

    Optic nerve (ON) damage following nonarteritic anterior ischemic optic neuropathy (NAION) and its models is associated with neurodegenerative inflammation. Minocycline is a tetracycline derivative antibiotic believed to exert a neuroprotective effect by selective alteration and activation of the neuroinflammatory response. We evaluated minocycline's post-induction ability to modify early and late post-ischemic inflammatory responses and its retinal ganglion cell (RGC)-neuroprotective ability. We used the rodent NAION (rNAION) model in male Sprague-Dawley rats. Animals received either vehicle or minocycline (33 mg/kg) daily intraperitoneally for 28 days. Early (3 days) ON-cytokine responses were evaluated, and oligodendrocyte death was temporally evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. Cellular inflammation was evaluated with immunohistochemistry, and RGC preservation was compared with stereology of Brn3a-positive cells in flat mounted retinas. Post-rNAION, oligodendrocytes exhibit a delayed pattern of apoptosis extending over a month, with extrinsic monocyte infiltration occurring only in the primary rNAION lesion and progressive distal microglial activation. Post-induction minocycline failed to improve retinal ganglion cell survival compared with the vehicle treated (893.14 vs. 920.72; p>0.9). Cytokine analysis of the rNAION lesion 3 days post-induction revealed that minocycline exert general inflammatory suppression without selective upregulation of cytokines associated with the proposed alternative or neuroprotective M2 inflammatory pathway. The pattern of cytokine release, extended temporal window of oligodendrocyte death, and progressive microglial activation suggests that selective neuroimmunomodulation, rather than general inflammatory suppression, may be required for effective repair strategies in ischemic optic neuropathies.

  8. Macrophage migration inhibitory factor (MIF) knockout preserves cardiac homeostasis through alleviating Akt-mediated myocardial autophagy suppression in high-fat diet-induced obesity.

    Science.gov (United States)

    Xu, X; Ren, J

    2015-03-01

    Macrophage migration inhibitory factor (MIF) has a role in the development of obesity and diabetes. However, whether MIF has a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high-fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy. Adult male wild-type (WT) and MIF knockout (MIF(-/-)) mice were placed on 45% high-fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio, locomotor activity and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using terminal dUTP nick end labeling staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF(-/-) mice were treated with recombinant mouse MIF (rmMIF). High-fat diet feeding elicited increased body weight gain, insulin resistance and caloric disturbance in WT and MIF(-/-) mice. High-fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF. These results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac

  9. Intratumoral distribution of CU-ATSM and FDG: immunohistochemical characterization of the region with high CU-ATSM or FDG accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Takako, Furukawa; Tsuneo, Saga; Yasuhisa, Fujibayashi [Institute of Radiological Sciences, Dept. of Diagnostic Imaging, Molecular Imaging Center, Nationa, Chiba (Japan); Takako, Furukawa; Takeshi, Tanaka; Yasuhisa, Fujibayashi [Fukui Univ., Biomedica Imaging Research Center (Japan)

    2006-07-01

    Intratumoral distribution of [Cu-64]Cu-di-acetyl-bis (N4-methyl-thio-semi-carbazone ) ({sup 64}Cu-ATSM) and fluorine-18 2-fluoro-2-deoxyglucose ({sup 18}FDG) in mice bearing tumors of four different origins, LLC1 (Lewis lung carcinoma), Meth-A (sarcoma), B16 (melanoma) and colon26 (adenocarcinoma), were compared to the immunohistochemical staining for proliferating cells (Ki67), blood vessels (CD34 or von Willebrand Factor (vWF)) and apoptotic cells (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method). With all the cell lines, {sup 64}Cu-ATSM and {sup 18}FDG were distributed to different regions of the tumor mass. The immunohistochemical study demonstrated that the high {sup 64}Cu-ASTM uptake regions were hypo-vascular and consisted of tumor cells arrested in cell cycle, while the high {sup 18}FGD uptake regions were hyper-vascular and consisted of proliferating cells. Through our study, it was revealed that one tumor mass contains two regions of different characteristics, which can be distinguished by {sup 64}Cu-ATSM and {sup 18}FDG. Since hypoxia and cell cycle arrest are critical factors to reduce the sensitivity of tumor to radiation and conventional chemotherapy, regions with such characteristics in tumor should be treated intensively as one of the primary targets. {sup 64}Cu-ATSM which can delineate hypoxic and cell cycle arrested regions in tumors can provide valuable information for cancer treatment as well as the possibility to treat such regions directly as an internal radiotherapy reagent. (author)

  10. [Establishment of a D-galactosamine/lipopolysaccharide induced acute-on-chronic liver failure model in rats].

    Science.gov (United States)

    Liu, Xu-hua; Chen, Yu; Wang, Tai-ling; Lu, Jun; Zhang, Li-jie; Song, Chen-zhao; Zhang, Jing; Duan, Zhong-ping

    2007-10-01

    To establish a practical and reproducible animal model of human acute-on-chronic liver failure for further study of the pathophysiological mechanism of acute-on-chronic liver failure and for drug screening and evaluation in its treatment. Immunological hepatic fibrosis was induced by human serum albumin in Wistar rats. In rats with early-stage cirrhosis (fibrosis stage IV), D-galactosamine and lipopolysaccharide were administered. Mortality and survival time were recorded in 20 rats. Ten rats were sacrificed at 4, 8, and 12 hours. Liver function tests and plasma cytokine levels were measured after D-galactosamine/lipopolysaccharide administration and liver pathology was studied. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Most of the rats treated with human albumin developed cirrhosis and fibrosis, and 90% of them died from acute liver failure after administration of D-galactosamine/lipopolysaccharide, with a mean survival time of (16.1+/-3.7) hours. Liver histopathology showed massive or submassive necrosis of the regenerated nodules, while fibrosis septa were intact. Liver function tests were compatible with massive necrosis of hepatocytes. Plasma level of TNFalpha increased significantly, parallel with the degree of the hepatocytes apoptosis. Plasma IL-10 levels increased similarly as seen in patients with acute-on-chronic liver failure. We established an animal model of acute-on-chronic liver failure by treating rats with human serum albumin and later with D-galactosamine and lipopolysaccharide. TNFalpha-mediated liver cell apoptoses plays a very important role in the pathogenesis of acute liver failure.

  11. Regulatory effect of Bcl-2 in ultraviolet radiation-induced apoptosis of the mouse crystalline lens.

    Science.gov (United States)

    Dong, Yuchen; Zheng, Yajuan; Xiao, Jun; Zhu, Chao; Zhao, Meisheng

    2016-03-01

    The aim of the present study was to analyze the role of Bcl-2 during the process of apoptosis in the mouse crystalline lens. In total, 12 normal mice served as the control group and 12 Bcl-2 knockout (K.O) mice served as the experimental group. The mouse crystalline lens was sampled for the detection of Bcl-2 and caspase-3 expression following exposure to ultraviolet (UV) radiation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine Bcl-2 expression in the groups of normal mice receiving UV radiation or not receiving UV radiation. Samples of the murine crystalline lens were microscopically harvested and analyzed using western blotting. Apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, caspase 3 activity was examined using enzyme-linked immunosorbent assay kits, and RT-qPCR was used to analyze caspase-3 expression levels. The results of the present study demonstrated that there was no statistically significant difference in the level of Bcl-2 gene transcription between the two groups. In addition, UV radiation did not change the macrostructure of the crystalline lens in the group of normal mice or the group of Bcl-2 K.O mice. The results of the TUNEL assay indicated that the normal-UV group exhibited a more significant apoptosis level compared with the Bcl-2 K.O-UV group. Furthermore, the mRNA expression level of caspase-3 in the normal-UV group was significantly higher compared with the normal-nonUV group (Plens.

  12. Cytotoxic effect of γ-sitosterol from Kejibeling (Strobilanthes crispus and its mechanism of action towards c-myc gene expression and apoptotic pathway

    Directory of Open Access Journals (Sweden)

    Susi Endrini

    2015-01-01

    Full Text Available Background: This study aimed to analyze the cytotoxicity effect of γ-sitosterol isolated from “Kejibeling” (Strobilanthes crispus, a medicinal plant, on several cancer cell lines. The mechanisms of the effects were studied through the expression of cancer-caused gene, c-myc and apoptotic pathways.Methods: This in vitro study was done using human colon cancer cell lines (Caco-2, liver cancer cell lines (HepG2, hormone-dependent breast cancer cell lines (MCF-7 and the normal liver cell lines (Chang Liver. The cytotoxic effect was measured through MTT assay and the potential cytotoxic value was calculated by determining the toxic concentration which may kill up to 50% of the total cell used (IC50. Meanwhile, the cytotoxic mechanism was studied by determining the effect of adding γ-sitosterol to the c-myc gene expression by reverse transciptase-polymerase chain reaction (RT-PCR. The effect of γ-sitosterol through apoptotic pathway was studied by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay.Results: γ-sitosterol was cytotoxic against Caco-2, HepG2, and MCF-7 with IC50-values of 8.3, 21.8, and 28.8 μg/mL, respectively. There were no IC50-values obtained from this compound against Chang Liver cell line. This compound induced apotosis on Caco-2 and HepG2 cell lines and suppressed the c-myc genes expression in both cells.Conclusion: γ-sitosterol was cytotoxic against colon and liver cancer cell lines and the effect was mediated by down-regulation of c-myc expression and induction of the apoptotic pathways.

  13. 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.

    2012-01-01

    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. PMID:22052013

  14. The effects of systemic, topical, and intralesional steroid treatments on apoptosis level of nasal polyps.

    Science.gov (United States)

    Kapucu, Burak; Cekin, Engin; Erkul, Bulent Evren; Cincik, Hakan; Gungor, Atila; Berber, Ufuk

    2012-09-01

    The purpose of this study was to compare the apoptotic responses to systemic, topical, and intrapolyp injection of glucocorticoid with no treatment in nasal polyps. Prospective, randomized controlled study. Tertiary training hospital. The study was performed on 48 patients with nasal polyposis in the Department of Otorhinolaryngology between 2008 and 2009. Patients were assigned to 1 of 4 groups of 12 patients. Group A was treated with oral methylprednisolone 1 mg/kg/d, and the dose was tapered gradually. Group B received 0.3 mL triamcinolone acetonide (40 mg/mL), which was injected into polyp tissue. Group C was treated with topical 55 µg triamcinolone acetonide 2 times daily for 1 month. Group D received no medication. Samples were collected endoscopically after the seventh day for groups A and B, the first month for group C, and the first visit for group D. Apoptotic indexes were determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Statistically significant differences in apoptotic index were found between each steroid-medicated group and the control group (P (D-A) = .0001; P (D-B) = .003; P (D-C) = .026) and between groups A and C (P (A-C) = .012). Group B did not differ significantly from either group A or C (P (A-B) = .11; P (B-C) = .75). The apoptotic index in nasal polyps treated with systemic, topical, and intrapolyp injection forms of glucocorticoids was higher than that in the control group. Systemic steroid treatment induced the most apoptosis.

  15. Beneficial effects of enteral nutrition containing with hydrolyzed whey peptide on warm ischemia/reperfusion injury in the rat liver.

    Science.gov (United States)

    Hanaoka, Jun; Shimada, Mitsuo; Utsunomiya, Toru; Morine, Yuji; Imura, Satoru; Ikemoto, Tetsuya; Mori, Hiroki; Sugimoto, Koji; Saito, Yu; Yamada, Shinichiro; Asanoma, Michihito

    2014-01-01

    This study examined the efficacy of enteral nutrition containing hydrolyzed whey peptide (HWP) on warm ischemia/reperfusion (I/R) injury in the rat liver. Male Wistar rats were subjected to 30 min of warm hepatic ischemia followed by immediate p.o. intake of enteral nutrition with WHP (HWP group) or 20% glucose solution (control group) (0.025 mL/g). The animals were killed at 6 or 12 h after reperfusion. The serum aspartate aminotransferase (AST) and alanine aminotransferase alt (ALT) levels were measured. The necrotic areas were assessed histologically. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and caspase-3 activation were assessed to evaluate apoptosis. The expressions of hepatic tumor necrosis factor (TNF)-α, interleukin (IL)-6 and nuclear factor (NF)-κB in the liver tissue were assessed by real time reverse transcription polymerase chain reaction. Significant reductions in the serum AST and ALT levels were seen in the HWP group compared with the control group at both 6 and 12 h after reperfusion. The necrotic areas and numbers of TUNEL positive cells were significantly decreased in the HWP group at 6 and 12 h after reperfusion. The caspase-3/7 activities were significantly decreased in HWP group at 6 and 12 h after reperfusion. The mRNA expressions of TNF-α and IL-6 were significantly reduced in the HWP group at 12 h after reperfusion. NF-κB mRNA expression was significantly increased in the HWP group at 6 and 12 h after reperfusion. Enteral nutrition containing HWP ameliorated the hepatic warm I/R injury possibly through the suppression of pro-inflammatory cytokine expressions and the induction of NF-κB in the rat liver. © 2013 The Japan Society of Hepatology.

  16. Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons

    Science.gov (United States)

    Wang, Wei-Ping; Iyo, Abiye H.; Miguel-Hidalgo, Javier; Regunathan, Soundar; Zhu, Meng-Yang

    2010-01-01

    Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-d-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, β-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 µM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property. PMID:16546145

  17. Positron emission tomography imaging of cardiomyocyte apoptosis with a novel molecule probe [18F]FP-DPAZn2

    Science.gov (United States)

    Sun, Ting; Tang, Ganghua; Tian, Hua; Hu, Kongzhen; Yao, Shaobo; Su, Yifan; Wang, Changqian

    2015-01-01

    Cardiomyocyte apoptosis plays a causal role in the development and progression of heart failure. Currently, there is no effective imaging agent that can be used to detect cardiomyocyte apoptosis in vivo. To target phosphatidylserine (PS) on the surface of the dying cell, we synthesized a novel 18F-labeled Zn2+-dipicolylamine (DPA) analog, [18F]FP-DPAZn2, and evaluated it for noninvasive imaging of cardiomyocyte apoptosis. In vitro, the fluorescence imaging of dansyl-DPAZn2 was suitable for detecting cardiomyocyte apoptosis, which was confirmed by confocal immunofluorescence imaging, terminal dUTP nick-end labeling (TUNEL) assay, and western blot assay. The in vivo biodistribution showed that the uptake ratios of [18F]FP-DPAZn2 in the heart were 4.41±0.29% ID/g at 5 min, 2.40 ± 0.43% ID/g at 30 min, 1.63 ± 0.26% ID/g at 60 min, and 1.43% ± 0.07 ID/g at 120 min post-injection. In vivo, the [18F]FP-DPAZn2 PET images showed more cardiac accumulation of radioactivity 60 min post-injection in acute myocardial infarction (AMI) rats than in normal rats, which was consistent with the findings of a histological analysis of the rat cardiac tissues in vitro. [18F]FP-DPAZn2 PET imaging has the capability for myocardial apoptosis detection, but the method will require improved myocardial uptake for the noninvasive evaluation of cardiomyocyte apoptosis in clinical settings. PMID:26416423

  18. A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold.

    Science.gov (United States)

    Luyckx, Valérie; Dolmans, Marie-Madeleine; Vanacker, Julie; Legat, Camille; Fortuño Moya, Cristina; Donnez, Jacques; Amorim, Christiani Andrade

    2014-04-01

    To create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot benefit from transplantation of cryopreserved ovarian tissue due to the threat of reintroducing malignant cells. In vivo experimental study. Gynecology research unit in a university hospital. Six-week-old female NMRI mice. Autografting of isolated preantral follicles and ovarian cells (OCs) encapsulated in two fibrin matrices containing low concentrations of fibrinogen (F; mg/mL) and thrombin (T; IU/mL): F12.5/T1 and F25/T4. Follicular density and development, OC survival and proliferation, inflammatory response, and vascularization. After 1 week, the follicle recovery rate ranged from 30.8% (F25/T4) to 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and at the growing stage (primary, secondary, and antral follicles), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by <1% apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations, and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in both F25/T4 and F12.5/T1 combinations. The present study is the first to show survival and growth of isolated murine ovarian follicles 1 week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  19. Bisphenol A induces spermatocyte apoptosis in rare minnow Gobiocypris rarus

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingying; Cheng, Mengqian; Wu, Lang; Zhang, Guo; Wang, Zaizhao, E-mail: zzwang@nwsuaf.edu.cn

    2016-10-15

    Highlights: • Adult male G. rarus were exposed to 225 μg/L BPA for 7, 21 and 63 days. • BPA could induce spermatocyte apoptosis in rare minnow testis. • The mitochondrial apoptotic pathway participated in the germ cell apoptosis. • The spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest. - Abstract: Bisphenol A (BPA) is an endocrine disruptor, and could induce germ cells apoptosis in the testis of mammals. But whether it could affect fish in the same mechanism has not’ been studied till now. In the present study, to investigate the influence of BPA on testis germ cells in fish, adult male rare minnow Gobiocypris rarus were exposed to 225 μg L{sup −1} (0.99 μM) BPA for 1, 3 and 9 weeks. Through TdT-mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM) analysis, we found that the amount of apoptotic spermatocytes significantly increased in a time dependent manner following BPA exposure. Western Blot results showed that the ratio of Bcl2/Bax, the important apoptosis regulators in intrinsic mitochondrial apoptotic pathway, was significantly decreased. qPCR showed that mRNA expression of several genes in mitochondrial apoptotic pathway including bcl2, bax, casp9, cytc and mcl1b were significantly changed following BPA exposure. In addition, mRNA expression of meiosis regulation genes (kpna7 and wee2), and genes involved in both apoptosis and meiosis (birc5, ccna1, and gsa1a) were also affected by BPA. Taken together, the present study demonstrated that BPA could induce spermatocytes apoptosis in rare minnow testis, and the apoptosis was probably under regulation of intrinsic mitochondrial apoptotic pathway. Moreover, the spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest.

  20. Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR

    International Nuclear Information System (INIS)

    Kijima, Kazuyasu; Toyosawa, Kaoru; Yasuba, Masashi; Matsuoka, Nobuo; Adachi, Tetsuya; Komiyama, Masatoshi; Mori, Chisato

    2004-01-01

    To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment

  1. Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling.

    Directory of Open Access Journals (Sweden)

    Ting-Ting Xiao

    Full Text Available Accumulating evidence indicates that oxymatrine (OMT possesses variously pharmacological properties, especially on the cardiovascular system. We previously demonstrated that activated calpain/apoptosis-inducing factor (AIF-mediated pathway was the key molecular mechanism in aldosterone (ALD induces cardiomyocytes apoptosis. In the present study, we extended the experimentation by investigating the effect of OMT on cardiomyocytes exposed to ALD, as compared to spironolactone (Spiro, a classical ALD receptor antagonist. Cardiomyocytes were pre-incubated with OMT, Spiro or vehicle for 1 h, and then, cardiomyocytes were exposed to ALD 24 h. The cell injury was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and lactate dehydrogenase (LDH leakage ratio. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, annexin V/PI staining, and relative caspase-3 activity assay. Furthermore, expression of pro-apoptotic proteins including truncated Bid (tBid, calpain and AIF were evaluated by western blot analysis. ALD stimulation increased cardiomyocytes apoptosis, caspase-3 activity and protein expression of calpain, tBid and AIF in the cytosol (p<0.05. Pre-incubated with cardiomyocytes injury and increased caspase-3 activity were significantly attenuated (p<0.05. Furthermore, OMT suppressed ALD-induced high expression of calpain and AIF. And these effects of OMT could be comparable to Spiro. These findings indicated that OMT might be a potential cardioprotective-agent against excessive ALD-induced cardiotoxicity, at least in part, mediated through inhibition of calpain/AIF signaling.

  2. 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. © 2015 The Royal Entomological Society.

  3. Lack of TXNIP protects against mitochondria-mediated apoptosis but not against fatty acid-induced ER stress-mediated beta-cell death.

    Science.gov (United States)

    Chen, Junqin; Fontes, Ghislaine; Saxena, Geetu; Poitout, Vincent; Shalev, Anath

    2010-02-01

    We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced beta-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects beta-cells against lipoapoptosis. RESARCH DESIGN AND METHODS: To determine the effects of fatty acids on beta-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced beta-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. Our results demonstrate that unlike glucose, fatty acids do not induce beta-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying beta-cell glucotoxicity, whereas it has very few protective effects against ER stress-mediated lipoapoptosis.

  4. Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

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    Zhao Ying-Zheng

    2010-11-01

    Full Text Available Abstract Background Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Methods Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Results Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4 hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Conclusion Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.

  5. Does sucralfate prevent apoptosis occurring in the ischemia/reperfusion-induced intestinal injury?

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    Sencan, A; Yilmaz, O; Ozer, E; Günşar, C; Genç, K; Ulukuş, C; Taneli, C; Mir, E

    2003-08-01

    We have shown in a previous study that sucralfate is beneficial in the prophylaxis and treatment of hypoxia/reoxygenation-induced intestinal injury. The aim of this study is to investigate whether sucralfate has any effect on the prevention of apoptosis in the ischemia/reperfusion (I/R)-induced intestinal injury. Rats were randomized into three groups. Group 1 and 2 were subjected to I/R. Group 1 (treatment group) received sucralfate while group 2 (treatment control group) did not. Group 3 served as a normal control group (sham group). The terminal ileum was harvested for histopathologic investigation by light microscopy. The presence of apoptotic enterocytes (DNA fragmentation in cell nuclei) was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) reaction. In treatment control group, 3 of 7 rats had severe inflammation. None of the sucralfate-treated rats showed severe inflammation, 6 of them only showed mild inflammatory changes (p < 0.05). The apoptotic percentage was found to be 37.1 +/- 9.4 in the sucralfate-treated group (group 1), whereas it was 45.4 +/- 3.9 in the untreated group (group 2) (p < 0.05). The sham group had a completely normal intestinal architecture. The present study shows that 1) the experimental model of I/R-induced intestinal injury induces enterocyte apoptosis; 2) sucralfate decreases enterocyte apoptosis in the experimental model of I/R-induced intestinal injury which may play a key role in the pathophysiological events leading to failure of the intrinsic gut barrier defense mechanisms.

  6. Increased intrahepatic apoptosis but reduced immune activation in HIV-HBV co-infected patients with advanced immunosuppression.

    Science.gov (United States)

    Iser, David M; Avihingsanon, Anchalee; Wisedopas, Naruemon; Thompson, Alexander J; Boyd, Alison; Matthews, Gail V; Locarnini, Stephen A; Slavin, John; Desmond, Paul V; Lewin, Sharon R

    2011-01-14

    to determine if intrahepatic immune activation is increased in HIV-hepatitis B virus (HBV) co-infected patients compared to HBV mono-infected patients and whether this reduced following HBV-active antiretroviral therapy (ART) in HIV-HBV co-infected patients. : Case-control observational study. we examined liver biopsies for markers of T-cell and monocyte infiltration and activation, natural killer cells, hepatic stellate cell (HSC) activation (staining for alpha smooth muscle actin) and apoptosis [using terminal dUTP nick-end labelling (TUNEL)] in treatment-naive Asian HIV-HBV co-infected (n = 16) and HBV mono-infected patients matched for age and HBV e-antigen status (n = 16). Liver biopsies from a subset of co-infected patients (n = 15) were also compared prior to and following 48 weeks of HBV-active ART. HIV-HBV co-infected patients had a median CD4 T-cell count of 25 cells/microl and lower alanine aminotransferase levels than HBV mono-infected patients (P = 0.03). In HIV-HBV co-infected patients, hepatocyte apoptosis was increased (P = 0.04) but there were fewer intrahepatic CD4 and CD8 T cells (P < 0.001), lower activation of intrahepatic T cells, Kupffer cells and HSC (P = 0.002, 0.008 and < 0.001, respectively). Following ART, there was a significant decrease in intrahepatic HBsAg staining (P = 0.04) and Kupffer cell activation (P = 0.003). we found no evidence of increased intrahepatic mononuclear and HSC activation in this cohort of HIV-HBV co-infected individuals with advanced immune suppression. An increase in intra-hepatic apoptosis in HIV-HBV co-infected individuals may potentially contribute to accelerated fibrosis in this setting. 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

  7. Apoptosis Governs the Elimination of Schistosoma japonicum from the Non-Permissive Host Microtus fortis

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    Peng, Jinbiao; Gobert, Geoffrey N.; Hong, Yang; Jiang, Weibin; Han, Hongxiao; McManus, Donald P.; Wang, Xinzhi; Liu, Jinming; Fu, Zhiqiang; Shi, Yaojun; Lin, Jiaojiao

    2011-01-01

    The reed vole, Microtus fortis, is the only known mammalian host in which schistosomes of Schistosoma japonicum are unable to mature and cause significant pathogenesis. However, little is known about how Schistosoma japonicum maturation (and, therefore, the development of schistosomiasis) is prevented in M. fortis. In the present study, the ultrastructure of 10 days post infection schistosomula from BALB/c mice and M. fortis were first compared using scanning electron microscopy and transmission electron microscopy. Electron microscopic investigations showed growth retardation and ultrastructural differences in the tegument and sub-tegumental tissues as well as in the parenchymal cells of schistosomula from M. fortis compared with those in BALB/c mice. Then, microarray analysis revealed significant differential expression between the schistosomula from the two rodents, with 3,293 down-regulated (by ≥2-fold) and 71 up-regulated (≥2 fold) genes in schistosomula from the former. The up-regulated genes included a proliferation-related gene encoding granulin (Grn) and tropomyosin. Genes that were down-regulated in schistosomula from M. fortis included apoptosis-inhibited genes encoding a baculoviral IAP repeat-containing protein (SjIAP) and cytokine-induced apoptosis inhibitor (SjCIAP), genes encoding molecules involved in insulin metabolism, long-chain fatty acid metabolism, signal transduction, the transforming growth factor (TGF) pathway, the Wnt pathway and in development. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and PI/Annexin V-FITC assays, caspase 3/7 activity analysis, and flow cytometry revealed that the percentages of early apoptotic and late apoptotic and/or necrotic cells, as well as the level of caspase activity, in schistosomula from M. fortis were all significantly higher than in those from BALB/c mice. PMID:21731652

  8. Protective effect of hexane and ethanol extract of piper longum L. On gentamicin-induced hair cell loss in neonatal cultures.

    Science.gov (United States)

    Yadav, Mukesh Kumar; Choi, June; Song, Jae-Jun

    2014-03-01

    Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Piper longum L. (PL) is a well-known spice and traditional medicine in Asia and Pacific islands, which has been reported to exhibit a wide spectrum of activity, including antioxidant activity. In this study, we evaluated the effect of hexane:ethanol (2:8) PL extract (subfraction of PL [SPL] extract) on GM-induced hair cell loss in basal, middle and apical regions in a neonatal cochlea cultures. The protective effects of SPL extract were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. The anti-apoptosis activity of SPL extract was measured using double labeling by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and myosin-7a staining. The radical-scavenging activity of SPL extract was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. SPL extract at a concentration of 1 µg/mL significantly inhibited GM-induced hair cell loss at basal and middle region of cochlea, while 5 µg/mL was effective against apical region hair cell loss. The protective effect of SPL extract was concentration dependent and hair cells retained their stereocilia in explants treated with SPL extract prior to treatment with 0.3 mM GM. SPL extract decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. The outer hair and inner hair counts were not decreased in SPL extract treated groups in compare to GM treated explants. Additionally, SPL extract showed concentration dependent radical scavenging activity in a DPPH assay. An anti-apoptosis effect and potent radical scavenger activity of SPL extract protects from GM-induced hair cell loss at basal, middle and apical regions in neonatal cochlea cultures.

  9. Mechanisms underlying the promotion of functional recovery by deferoxamine after spinal cord injury in rats

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    Jian Hao

    2017-01-01

    Full Text Available Deferoxamine, a clinically safe drug used for treating iron overload, also repairs spinal cord injury although the mechanism for this action remains unknown. Here, we determined whether deferoxamine was therapeutic in a rat model of spinal cord injury and explored potential mechanisms for this effect. Spinal cord injury was induced by impacting the spinal cord at the thoracic T10 vertebra level. One group of injured rats received deferoxamine, a second injured group received saline, and a third group was sham operated. Both 2 days and 2 weeks after spinal cord injury, total iron ion levels and protein expression levels of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1β and the pro-apoptotic protein caspase-3 in the spinal cords of the injured deferoxamine-treated rats were significantly lower than those in the injured saline-treated group. The percentage of the area positive for glial fibrillary acidic protein immunoreactivity and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were also significantly decreased both 2 days and 2 weeks post injury, while the number of NeuN-positive cells and the percentage of the area positive for the oligodendrocyte marker CNPase were increased in the injured deferoxamine-treated rats. At 14–56 days post injury, hind limb motor function in the deferoxamine-treated rats was superior to that in the saline-treated rats. These results suggest that deferoxamine decreases total iron ion, tumor necrosis factor-α, interleukin-1β, and caspase-3 expression levels after spinal cord injury and inhibits apoptosis and glial scar formation to promote motor function recovery.

  10. A chick model of retinal detachment: cone rich and novel.

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    Colleen M Cebulla

    Full Text Available Development of retinal detachment models in small animals can be difficult and expensive. Here we create and characterize a novel, cone-rich retinal detachment (RD model in the chick.Retinal detachments were created in chicks between postnatal days 7 and 21 by subretinal injections of either saline (SA or hyaluronic acid (HA. Injections were performed through a dilated pupil with observation via surgical microscope, using the fellow eye as a control. Immunohistochemical analyses were performed at days 1, 3, 7, 10 and 14 after retinal detachment to evaluate the cellular responses of photoreceptors, Müller glia, microglia and nonastrocytic inner retinal glia (NIRG. Cell proliferation was detected with bromodeoxyuridine (BrdU-incorporation and by the expression of proliferating cell nuclear antigen (PCNA. Cell death was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL. As in mammalian models of RD, there is shortening of photoreceptor outer segments and mis-trafficking of photoreceptor opsins in areas of RD. Photoreceptor cell death was maximal 1 day after RD, but continued until 14 days after RD. Müller glia up-regulated glial fibriliary acidic protein (GFAP, proliferated, showed interkinetic nuclear migration, and migrated to the subretinal space in areas of detachment. Microglia became reactive; they up-regulated CD45, acquired amoeboid morphology, and migrated toward outer retina in areas of RD. Reactive NIRG cells accumulated in detached areas.Subretinal injections of SA or HA in the chick eye successfully produced retinal detachments and cellular responses similar to those seen in standard mammalian models. Given the relatively large eye size, and considering the low cost, the chick model of RD offers advantages for high-throughput studies.

  11. Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation.

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    Ramírez, M Á; Pericuesta, E; Yáñez-Mó, M; Palasz, A; Gutiérrez-Adán, A

    2011-02-01

    Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. After 3 days culture or after long-term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT-mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long-term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long-term culture of mES cells in vitro. © 2010 Blackwell Publishing Ltd.

  12. Protective effects of seabuckthorn pulp and seed oils against radiation-induced acute intestinal injury

    International Nuclear Information System (INIS)

    Shi, Jing; Wang, Lan; Lu, Yan; Ji, Yue; Wang, Yaqing; Dong, Ke; Kong, Xiangqing; Sun, Wei

    2017-01-01

    Radiation-induced gastrointestinal syndrome, including nausea, diarrhea and dehydration, contributes to morbidity and mortality after medical or industrial radiation exposure. No safe and effective radiation countermeasure has been approved for clinical therapy. In this study, we aimed to investigate the potential protective effects of seabuckthorn pulp and seed oils against radiation-induced acute intestinal injury. C57/BL6 mice were orally administered seabuckthorn pulp oil, seed oil and control olive oil once per day for 7 days before exposure to total-body X-ray irradiation of 7.5 Gy. Terminal deoxynucleotidyl transferase dUTP nick end labeling, quantitative real-time polymerase chain reaction and western blotting were used for the measurement of apoptotic cells and proteins, inflammation factors and mitogen-activated protein (MAP) kinases. Seabuckthorn oil pretreatment increased the post-radiation survival rate and reduced the damage area of the small intestine villi. Both the pulp and seed oil treatment significantly decreased the apoptotic cell numbers and cleaved caspase 3 expression. Seabuckthorn oil downregulated the mRNA level of inflammatory factors, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-8. Both the pulp and seed oils elevated the level of phosphorylated extracellular-signal-regulated kinase and reduced the levels of phosphorylated c-Jun N-terminal kinase and p38. Palmitoleic acid (PLA) and alpha linolenic acid (ALA) are the predominant components of pulp oil and seed oil, respectively. Pretreatment with PLA and ALA increased the post-radiation survival time. In conclusion, seabuckthorn pulp and seed oils protect against mouse intestinal injury from high-dose radiation by reducing cell apoptosis and inflammation. ALA and PLA are promising natural radiation countermeasure candidates.

  13. Expression of hPNAS-4 Radiosensitizes Lewis Lung Cancer

    International Nuclear Information System (INIS)

    Zeng Hui; Yuan Zhu; Zhu Hong; Li Lei; Shi Huashan; Wang Zi; Fan Yu; Deng Qian; Zeng Jianshuang; He Yinbo; Xiao Jianghong; Li Zhiping

    2012-01-01

    Purpose: This study aimed to transfer the hPNAS-4 gene, a novel apoptosis-related human gene, into Lewis lung cancer (LL2) and observe its radiosensitive effect on radiation therapy in vitro and in vivo. Methods and Materials: The hPNAS-4 gene was transfected into LL2 cells, and its expression was detected via western blot. Colony formation assay and flow cytometry were used to detect the growth and apoptosis of cells treated with irradiation/PNAS-4 in vitro. The hPNAS-4 gene was transferred into LL2-bearing mice through tail vein injection of the liposome/gene complex. The tumor volumes were recorded after radiation therapy. Proliferating cell nuclear antigen (PCNA) immunohistochemistry staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect the tumor cell growth and apoptosis in vivo. Results: The hPNAS-4 gene was successfully transferred into LL2 cells and tumor tissue, and its overexpressions were confirmed via western blot analysis. Compared with the control, empty plasmid, hPNAS-4, radiation, and empty plasmid plus radiation groups, the hPNAS-4 plus radiation group more significantly inhibited growth and enhanced apoptosis of LL2 cells in vitro and in vivo (P<.05). Conclusions: The hPNAS-4 gene was successfully transferred into LL2 cells and tumor tissue and was expressed in both LL2 cell and tumor tissue. The hPNAS-4 gene therapy significantly enhanced growth inhibition and apoptosis of LL2 tumor cells by radiation therapy in vitro and in vivo. Therefore, it may be a potential radiosensitive treatment of radiation therapy for lung cancer.

  14. Protection of neurons in the retinal ganglion cell layer against excitotoxicity by the N-acylethanolamine, N-linoleoylethanolamine

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    Duncan RS

    2011-04-01

    Full Text Available R. Scott Duncan1,*, Hua Xin1,*, Daryl L Goad1, Kent D Chapman2,3, Peter Koulen1,31Vision Research Center and Departments of Ophthalmology and Basic Medical Science, School of Medicine, University of Missouri, Kansas City, MO, USA; 2Department of Biological Sciences, University of North Texas, Denton, TX, USA; 3Center for Plant Lipid Research, University of North Texas, Denton, TX, USA *Authors contributed equallyAbstract: Retinal ganglion cell (RGC death is a hallmark of neurodegenerative diseases and disease processes of the eye, including glaucoma. The protection of RGCs has been an important strategy for combating glaucoma, but little clinical success has been reported to date. One pathophysiological consequence of glaucoma is excessive extracellular glutamate subsequently leading to excitotoxicity in the retina. Endocannabinoids, such as the N-acylethanolamine (NAE, arachidonylethanolamine (NAE 20:4, exhibit neuroprotective properties in some models of neurodegenerative disease. The majority of NAEs, however, are not cannabinoids, and their physiological function is not clear. Here, we determined whether the noncannabinoid NAE, linoleoylethanolamine (NAE18:2, protects neurons in the RGC layer against glutamate excitotoxicity in ex-vivo retina cultures. Using a terminal deoxynucleotidyl transferase-mediated dUTP (2´-deoxyuridine 5´-triphosphate nick-end labeling (TUNEL assay, we determined that NAE18:2 reduces the number of apoptotic RGC layer neurons in response to glutamate and conclude that NAE18:2 is a neuroprotective compound with potential for treating glaucomatous retinopathy.Keywords: neuroprotection, glutamate, calcium signaling, immunocytochemistry, eye, vision, glaucoma.

  15. Effects of parenteral papaverine and piracetam administration on cochlea following acoustic trauma.

    Science.gov (United States)

    Kum, Nurcan Yurtsever; Yilmaz, Yavuz F; Gurgen, Seren G; Kum, Rauf O; Ozcan, Muge; Unal, Adnan

    2018-01-01

    Noise exposure, the main cause of hearing loss in countries with lot of industries, may result both in temporary or permanent hearing loss. The goal of this study was to investigate the effects of parenteral papaverine and piracetam administration following an acoustic trauma on hearing function with histopathologic correlation. Eighteen Wistar albino rats exposed to noise for 8 h in a free environment were included. We divided the study population into three groups, and performed daily intraperitoneal injections of papaverine, piracetam, and saline, respectively, throughout the study. We investigated the histopathologic effects of cellular apoptosis on inner hair cells (IHCs) and outer hair cells (OHCs) and compared the distortion product otoacoustic emissions (DPOAEs) thresholds among the groups. On the 3 rd and 7 th days, DPOAE thresholds at 8 kHz were significantly higher both in papaverine and piracetam groups compared with the control group (P = 0.004 for 3 rd day, P = 0.016 and P = 0.028 for 7 th day, respectively). On the 14 th day, piracetam group had significantly higher mean thresholds at 8 kHz (P = 0.029); however, papaverine group had similar mean thresholds compared to the control group (P = 0.200). On the 3 rd and 7 th days following acoustic trauma, both IHC and OHC loss were significantly lower in both papaverine and piracetam groups. On the 7 th day, the mean amount of apoptotic IHCs and OHCs identified using Caspase-3 method were significantly lower in both groups, but the mean amount identified using terminal deoxynucleotidyl transferase dUTP nick end labeling method were similar in both groups compared to the control group. We demonstrated the effects of papaverine and piracetam on the recovery of cochlear damage due to acoustic trauma on experimental animals using histopathologic and electrophysiologic examinations.

  16. Urtica dioica dichloromethane extract induce apoptosis from intrinsic pathway on human prostate cancer cells (PC3).

    Science.gov (United States)

    Mohammadi, A; Mansoori, B; Aghapour, M; Baradaran, B

    2016-03-31

    Prostate cancer is considered as the major cause of death among men around the world. There are a number of medicinal plants triggering apoptosis response in cancer cells, thus have a therapeutic potential. Therefore, further studies to characterize beneficial properties of these plants in order to introduce novel anti-cancer drugs are the interest of recent researches on the alternative medicine. On the other hand, due to traditional uses and availability of Urtica dioica extract, we decided to evaluate the efficacy of this medicinal herb on pc3 prostate cancer cell line. In the present study the cytotoxic effects of Urtica dioica extract were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and trypan blue viability dye. Then, DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were exploited to measure cell death and apoptosis stage. The expression levels of caspase 3, caspase 9 and Bcl-2 genes were quantified by Real-Time PCR. Finally, Cell cycle was analyzed by flow cytometry. MTT assay showed that dichloromethanolic extract of Urtica dioica significantly inhibited the cell growth. According to the DNA fragmentation and TUNEL assay results, the herbal extract was able to induce apoptosis in prostate cancer cells. Our findings also demonstrated that the plant extract substantially increases the caspase 3 and 9 mRNA expression, while decreases Bcl-2. Cell cycle arrest was occurred in G2 stage, due to the results of flow cytometry. These results indicate that dichloromethanolic extract of Urtica dioica can successfully induce apoptosis in PC3 cells. Therefore, it could be used as a novel therapeutic candidate for prostate tumor treatment.

  17. The Protective Effect of Cordycepin On Alcohol-Induced Osteonecrosis of the Femoral Head

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    Yi-Xuan Chen

    2017-08-01

    Full Text Available Background: Alcohol abuse is known to be a leading risk factor for atraumatic osteonecrosis of the femoral head (ONFH, in which the suppression of osteogenesis plays a critical role. Cordycepin benefits bone metabolism; however, there has been no study to determine its effect on osteonecrosis. Methods: Human bone mesenchymal stem cells (hBMSCs were identified by multi-lineage differentiation. Alkaline phosphatase (ALP activity, RT-PCR, western blots, immunofluorescent assay and Alizarin red staining of BMSCs were evaluated. A rat model of alcohol-induced ONFH was established to investigate the protective role of cordycepin against ethanol. Hematoxylin & eosin (H&E staining and micro-computerized tomography (micro-CT were performed to observe ONFH. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL. Immunohistochemical staining was carried out to detect OCN and COL1. Results: Ethanol significantly suppressed ALP activity, decreased gene expression of OCN and BMP2, lowered levels of RUNX2 protein, and reduced immunofluorescence staining of OCN and COL1 and calcium formation of hBMSCs. However, these inhibitory effects were attenuated by cordycepin co-treatment at concentrations of 1 and 10 µg/mL Moreover, it was revealed that the osteo-protective effect of cordycepin was associated with modulation of the Wnt/β-catenin pathway. In vivo, by micro-CT, TUNEL and immunohistochemical staining of OCN and COL1, we found that cordycepin administration prevented alcohol-induced ONFH. Conclusion: Cordycepin treatment to enhance osteogenesis may be considered a potential therapeutic approach to prevent the development of alcohol-induced ONFH.

  18. Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

    Science.gov (United States)

    Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

    2004-12-01

    In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

  19. Treatment of Arsenite Intoxication-Induced Peripheral Vasculopathy with Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yi-Hung Chiang

    2018-03-01

    Full Text Available Arsenite (As, a notorious toxic metal, is ubiquitously distributed in the earth and poses a serious threat to human health. Histopathological lesions of As intoxication are known as thromboangiitis obliterans, which are resistant to current treatment and often lead to lower limb amputation. In this study, we attempt to find that treatment with mesenchymal stem cells (MSCs may be effective for As-induced vasculopathy. We first conducted an in vitro study with a co-culture system containing human MSCs and human umbilical vein endothelial cells (HUVECs and treated individual and co-cultured cells with various concentrations of arsenite. We also designed an in vivo study in which Sprague Dawley (SD rats received periodic intraperitoneal (IP injections of 16 ppm arsenite for 12 weeks. MSCs were harvested from BALB/c mice that were transplanted via tail vein injection. We found that there was significantly higher cellular viability in human mesenchymal stem cells (hMSCs than in HUVECs under concentrations of arsenite between 15 and 25 μM. The Annexin V apoptosis assay further confirmed this finding. Cytokine array assay for As-conditioned media revealed an elevated vascular endothelial growth factor (VEGF level secreted by MSCs, which is crucial for HUVEC survival and was evaluated by an siRNA VEGF knockdown test. In the in vivo study, we demonstrated early apoptotic changes in the anterior tibial vessels of As-injected SD rats with a Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay, but these apoptotic changes were less frequently observed upon MSCs transplantation, indicating that the cytoprotective effect of MSCs successfully protected against As-induced peripheral vasculopathy. The feasibility of MSCs to treat and /or prevent the progression of As-induced vasculopathy is justified. Further clinical studies are required to demonstrate the therapeutic efficacy of MSCs in patients suffering from As intoxication with

  20. The effects of heavy ion particles on the developing murine cerebellum, with special reference to cell death

    International Nuclear Information System (INIS)

    Kinoshita, Chikako; Yaoi, Takeshi; Fushiki, Shinji; Nojima, Kumie

    2003-01-01

    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-μm-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)

  1. Tumor Response and Apoptosis of N1-S1 Rodent Hepatomas in Response to Intra-arterial and Intravenous Benzamide Riboside

    International Nuclear Information System (INIS)

    McLennan, Gordon; Bennett, Stacy L.; Ju, Shenghong; Babsky, Andriy; Bansal, Navin; Shorten, Michelle L.; Levitin, Seth; Bonnac, Laurent; Panciewicz, Krystoff W.; Jayaram, Hiramagular N.

    2012-01-01

    Purpose: Benzamide riboside (BR) induces tumor apoptosis in multiple cell lines and animals. This pilot study compares apoptosis and tumor response in rat hepatomas treated with hepatic arterial BR (IA) or intravenous (IV) BR. Methods: A total of 10 6 N1-S1 cells were placed in the left hepatic lobes of 15 Sprague-Dawley rats. After 2 weeks, BR (20 mg/kg) was infused IA (n = 5) or IV (n = 5). One animal in each group was excluded for technical factors, which prevented a full dose administration (1 IA and 1 IV). Five rats received saline (3 IA and 2 IV). Animals were killed after 3 weeks. Tumor volumes after IA and IV treatments were analyzed by Wilcoxon rank sum test. The percentage of tumor and normal liver apoptosis was counted by using 10 fields of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-stained slides at 40× magnification. The percentage of apoptosis was compared between IV and IA administrations and with saline sham-treated rats by the Wilcoxon rank sum test. Results: Tumors were smaller after IA treatment, but this did not reach statistical significance (0.14 IA vs. 0.57 IV; P = 0.138). There was much variability in percentage of apoptosis and no significant difference between IA and IV BR (44.49 vs. 1.52%; P = 0.18); IA BR and saline (44.49 vs. 33.83%; P = 0.66); or IV BR and saline (1.52 vs. 193%; P = 0.18). Conclusions: Although differences in tumor volumes did not reach statistical significance, there was a trend toward smaller tumors after IA BR than IV BR in this small pilot study. Comparisons of these treatment methods will require a larger sample size and repeat experimentation.

  2. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Pulsed electric field increases reproduction.

    Science.gov (United States)

    Panagopoulos, Dimitris J

    2016-01-01

    Purpose To study the effect of pulsed electric field - applied in corona discharge photography - on Drosophila melanogaster reproduction, possible induction of DNA fragmentation, and morphological alterations in the gonads. Materials and methods Animals were exposed to different field intensities (100, 200, 300, and 400 kV/m) during the first 2-5 days of their adult lives, and the effect on reproductive capacity was assessed. DNA fragmentation during early- and mid-oogenesis was investigated by application of the TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) assay. Sections of follicles after fixation and embedding in resins were observed for possible morphological/developmental abnormalities. Results The field increased reproduction by up to 30% by increasing reproductive capacity in both sexes. The effect increased with increasing field intensities. The rate of increase diminished at the strongest intensities. Slight induction of DNA fragmentation was observed exclusively in the nurse (predominantly) and follicle cells, and exclusively at the two most sensitive developmental stages, i.e., germarium and predominantly stage 7-8. Sections of follicles from exposed females at stages of early and mid-oogennesis other than germarium and stages 7-8 did not reveal abnormalities. Conclusions (1) The specific type of electric field may represent a mild stress factor, inducing DNA fragmentation and cell death in a small percentage of gametes, triggering the reaction of the animal's reproductive system to increase the rate of gametogenesis in order to compensate the loss of a small number of gametes. (2) The nurse cells are the most sensitive from all three types of egg chamber cells. (3) The mid-oogenesis checkpoint (stage 7-8) is more sensitive to this field than the early oogenesis one (germarium) in contrast to microwave exposure. (4) Possible therapeutic applications, or applications in increasing fertility, should be investigated.

  4. Hepatitis E Virus Induces Hepatocyte Apoptosis via Mitochondrial Pathway in Mongolian Gerbils

    Directory of Open Access Journals (Sweden)

    Yifei Yang

    2018-03-01

    Full Text Available Previous studies demonstrated that Mongolian gerbils can be infected by hepatitis E virus (HEV, which induces the hepatic injury. Here, the mitochondria in hepatocytes from HEV-infected gerbils were considerably swollen, thin cristae. After HEV infection, the activity of superoxide dismutase significantly decreased (p < 0.01, while malondialdehyde concentrations significantly increased, compared with those in the control group (p < 0.01. Adenosine triphosphatase levels decreased significantly in the hepatocyte of the inoculated groups, compared with those in control group (p < 0.05 at days 21, 28, 42 post-inoculation (dpi as well. Furthermore, the levels of ATP synthetase ATP5A1 significantly decreased during HEV infection, compared with those in the control group (p < 0.05. According to the TdT mediated dUTP nick end labeling (TUNEL detection, TUNEL positive hepatocytes increased in the inoculated group, compared with that in the control group (p < 0.05. Up-regulation of the mitochondrion-mediated apoptosis regulating proteins, Bax and Bcl-2, in the HEV-infected gerbils (p < 0.05 was observed. However, cytochrome c levels in mitochondria decreased, while this molecule was detected in the cytoplasm of the infected animals, in contrast to that in the control group. Apaf-1, and active caspase-9 and -3 levels were shown to be significantly higher in the inoculated group compared with those in the control group (p < 0.05. Taken together, our results demonstrated that HEV infection induces hepatocyte injuries and activity of the mitochondrial apoptotic pathway, which trigger the hepatocyte apoptosis in Mongolian gerbils.

  5. 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.

  6. Induction of Heat Shock Protein 70 Ameliorates Ultraviolet-Induced Photokeratitis in Mice

    Directory of Open Access Journals (Sweden)

    Yukihiro Horie

    2013-01-01

    Full Text Available Acute ultraviolet (UV B exposure causes photokeratitis and induces apoptosis in corneal cells. Geranylgeranylacetone (GGA is an acyclic polyisoprenoid that induces expression of heat shock protein (HSP70, a soluble intracellular chaperone protein expressed in various tissues, protecting cells against stress conditions. We examined whether induction of HSP70 has therapeutic effects on UV-photokeratitis in mice. C57 BL/6 mice were divided into four groups, GGA-treated (500 mg/kg/mouse and UVB-exposed (400 mJ/cm2, GGA-untreated UVB-exposed (400 mJ/cm2, GGA-treated (500 mg/kg/mouse but not exposed and naive controls. Eyeballs were collected 24 h after irradiation, and corneas were stained with hematoxylin and eosin (H&E and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL. HSP70, reactive oxygen species (ROS production, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and protein kinase B (Akt expression were also evaluated. Irradiated corneal epithelium was significantly thicker in the eyes of mice treated with GGA compared with those given the vehicle alone (p < 0.01. Significantly fewer TUNEL-positive cells were observed in the eyes of GGA-treated mice than controls after irradiation (p < 0.01. Corneal HSP70 levels were significantly elevated in corneas of mice treated with GGA (p < 0.05. ROS signal was not affected by GGA. NF-κB activation was reduced but phospho-(Ser/Ther Akt substrate expression was increased in corneas after irradiation when treated with GGA. GGA-treatment induced HSP70 expression and ameliorated UV-induced corneal damage through the reduced NF-κB activation and possibly increased Akt phosphorilation.

  7. Insulin-Like growth factor-II (IGF-II) prevents proinflammatory cytokine-induced apoptosis and significantly improves islet survival after transplantation.

    Science.gov (United States)

    Hughes, Amy; Mohanasundaram, Daisy; Kireta, Svjetlana; Jessup, Claire F; Drogemuller, Chris J; Coates, P Toby H

    2013-03-15

    The early loss of functional islet mass (50-70%) due to apoptosis after clinical transplantation contributes to islet allograft failure. Insulin-like growth factor (IGF)-II is an antiapoptotic protein that is highly expressed in β-cells during development but rapidly decreases in postnatal life. We used an adenoviral (Ad) vector to overexpress IGF-II in isolated rat islets and investigated its antiapoptotic action against exogenous cytokines interleukin-1β- and interferon-γ-induced islet cell death in vitro. Using an immunocompromised marginal mass islet transplant model, the ability of Ad-IGF-II-transduced rat islets to restore euglycemia in nonobese diabetic/severe combined immunodeficient diabetic recipients was assessed. Ad-IGF-II transduction did not affect islet viability or function. Ad-IGF-II cytokine-treated islets exhibited decreased cell death (40% ± 2.8%) versus Ad-GFP and untransduced control islets (63.2% ± 2.5% and 53.6% ± 2.3%, respectively). Ad-IGF-II overexpression during cytokine treatment resulted in a marked reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (8.3% ± 1.4%) versus Ad-GFP control (41% ± 4.2%) and untransduced control islets (46.5% ± 6.2%). Western blot analysis confirmed that IGF-II inhibits apoptosis via activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. Transplantation of IGF-II overexpressing islets under the kidney capsule of diabetic mice restored euglycemia in 77.8% of recipients compared with 18.2% and 47.5% of Ad-GFP and untransduced control islet recipients, respectively (Pislet transplant outcomes in vivo. Antiapoptotic gene transfer is a potentially powerful tool to improve islet survival after transplantation.

  8. DJ-1 Modulates Nuclear Erythroid 2–Related Factor-2–Mediated Protection in Human Primary Alveolar Type II Cells in Smokers

    Science.gov (United States)

    Bahmed, Karim; Messier, Elise M.; Zhou, Wenbo; Tuder, Rubin M.; Freed, Curt R.; Chu, Hong Wei; Kelsen, Steven G.; Bowler, Russell P.; Mason, Robert J.

    2016-01-01

    Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2–related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases. PMID:27093578

  9. Differences in neural crest sensitivity to ethanol account for the infrequency of anterior segment defects in the eye compared with craniofacial anomalies in a zebrafish model of fetal alcohol syndrome.

    Science.gov (United States)

    Eason, Jessica; Williams, Antionette L; Chawla, Bahaar; Apsey, Christian; Bohnsack, Brenda L

    2017-09-01

    Ethanol (ETOH) exposure during pregnancy is associated with craniofacial and neurologic abnormalities, but infrequently disrupts the anterior segment of the eye. In these studies, we used zebrafish to investigate differences in the teratogenic effect of ETOH on craniofacial, periocular, and ocular neural crest. Zebrafish eye and neural crest development was analyzed by means of live imaging, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, immunostaining, detection of reactive oxygen species, and in situ hybridization. Our studies demonstrated that foxd3-positive neural crest cells in the periocular mesenchyme and developing eye were less sensitive to ETOH than sox10-positive craniofacial neural crest cells that form the pharyngeal arches and jaw. ETOH increased apoptosis in the retina, but did not affect survival of periocular and ocular neural crest cells. ETOH also did not increase reactive oxygen species within the eye. In contrast, ETOH increased ventral neural crest apoptosis and reactive oxygen species production in the facial mesenchyme. In the eye and craniofacial region, sod2 showed high levels of expression in the anterior segment and in the setting of Sod2 knockdown, low levels of ETOH decreased migration of foxd3-positive neural crest cells into the developing eye. However, ETOH had minimal effect on the periocular and ocular expression of transcription factors (pitx2 and foxc1) that regulate anterior segment development. Neural crest cells contributing to the anterior segment of the eye exhibit increased ability to withstand ETOH-induced oxidative stress and apoptosis. These studies explain the rarity of anterior segment dysgenesis despite the frequent craniofacial abnormalities in fetal alcohol syndrome. Birth Defects Research 109:1212-1227, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Insulin-like growth factor-1 (IGF-1) promotes primordial follicle growth and reduces DNA fragmentation through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling pathway.

    Science.gov (United States)

    Bezerra, Maria É S; Barberino, Ricássio S; Menezes, Vanúzia G; Gouveia, Bruna B; Macedo, Taís J S; Santos, Jamile M S; Monte, Alane P O; Barros, Vanessa R P; Matos, Maria H T

    2018-05-30

    We investigated the effects of insulin-like growth factor 1 (IGF-1) on the morphology and follicular activation of ovine preantral follicles cultured in situ and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is involved in IGF-1 action in the sheep ovary. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) analyses (fresh control) or cultured in supplemented alpha-minimum essential medium (α-MEM+; control) or α-MEM+ with IGF-1 (1, 10, 50, 100 or 200ngmL-1) for 7 days. Follicles were classified as normal or atretic, primordial or growing and the oocyte and follicle diameters were measured. DNA fragmentation was evaluated by TUNEL assay. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed on the fresh control, α-MEM+ and 100ngmL-1 IGF-1 samples. Inhibition of PI3K activity was performed through pretreatment with the PI3K inhibitor LY294002 and phosphorylated AKT (pAKT) expression was analysed after culture in the absence or presence of LY294002. IGF-1 at 100ngmL-1 increased (PIGF-1. LY294002 significantly inhibited follicular activation stimulated by α-MEM+ and 100ngmL-1 IGF-1 and reduced pAKT expression in follicles. Overall, IGF-1 at 100ngmL-1 promoted primordial follicle activation, cell proliferation and reduced DNA fragmentation after in situ culture through the PI3K/AKT pathway.

  11. Cycloheximide and actinomycin D delay death and affect bcl-2, bax, and Ice gene expression in astrocytes under in vitro ischemia.

    Science.gov (United States)

    Yu, Albert Cheung Hoi; Yung, Hon Wa; Hui, Michael Hung Kit; Lau, Lok Ting; Chen, Xiao Qian; Collins, Richard A

    2003-10-15

    An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (alpha and beta), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (alpha and beta) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (alpha and beta) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis. Copyright 2003 Wiley-Liss, Inc.

  12. Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway.

    Science.gov (United States)

    Zhang, Qin; Huang, Wei-Dong; Lv, Xue-Ying; Yang, Yun-Mei

    2012-05-01

    Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.

  13. Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

    Science.gov (United States)

    Pirger, Zsolt; Rácz, Boglárka; Kiss, Tibor

    2009-02-01

    PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

  14. Combining portable Raman probes with nanotubes for theranostic applications.

    Science.gov (United States)

    Bhirde, Ashwinkumar A; Liu, Gang; Jin, Albert; Iglesias-Bartolome, Ramiro; Sousa, Alioscka A; Leapman, Richard D; Gutkind, J Silvio; Lee, Seulki; Chen, Xiaoyuan

    2011-01-01

    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 using a simple

  15. Protective effects of astragaloside IV on db/db mice with diabetic retinopathy.

    Directory of Open Access Journals (Sweden)

    Yuzhi Ding

    Full Text Available Diabetic retinopathy (DR is a common diabetic eye disease which is well-known as the result of microvascular retinal changes. Although the potential biological functions of astragaloside IV (AS IV have long been described in traditional system of medicine, its protective effect on DR remains unclear. This study aims to investigate the function and mechanism of AS IV on type 2 diabetic db/db mice.Db/db mice were treated with AS IV (4.5 mg/kg or 9 mg/kg or physiological saline by oral gavage for 20 weeks along with db/m mice. In each group, retinal ganglion cell (RGC function was measured by pattern electroretinogram (ERG and apoptosis was determined by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining. Blood and retina aldose reductase (AR activity were quantified by chemiluminescence analysis. The expressions of phosporylated-ERK1/2, NF-κB were determined by Western blot analysis. Furthermore, the expression of related downstream proteins were quantified by Label-based Mouse Antibody Array.Administration of AS IV significantly improved the amplitude in pattern ERG and reduced the apoptosis of RGCs.in db/db mice. Furthermore, downregulation of AR activity, ERK1/2 phosphorylation, NF-κB and related cytokine were observed in AS IV treatment group.Our study indicated that AS IV, as an inhibitor of AR, could prevent the activation of ERK1/2 phosporylation and NF-kB and further relieve the RGCs disfunction in db/db mice with DR. It has provided a basis for investigating the clinical efficacy of AR inhibitors in preventing DR.

  16. α2-Adrenergic modulation of the glutamate receptor and transporter function in a chronic ocular hypertension model.

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    Jung, Kyoung In; Kim, Jie Hyun; Park, Chan Kee

    2015-10-15

    Excitotoxicity, glutamate-induced toxic effects to retinal ganglion cells (RGCs), is one of several mechanisms of RGC loss suggested in glaucoma. In this study, we focused on the role of glutamate transporter of glial cells as well as N-methyl-d-aspartate (NMDA) receptor with regard to glutamate toxicity in glaucoma. We also investigated whether α2-adrenoceptor activation could modulate glutamate transporters and NMDA receptors in a chronic ocular hypertension model. Brimonidine 0.15% was administered topically to the eyes of experimental glaucoma and control animals twice daily. After 8 weeks of intraocular pressure (IOP) elevation, staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) revealed an increase in the ganglion cell layer, and the number of TUNEL-positive cells was reduced by brimonidine treatment (P<0.05). Animals with experimentally induced glaucoma exhibited an increase in retinal stress marker glial fibrillary acidic protein (GFAP) immunoreactivity; brimonidine treatment reduced GFAP. Excitatory amino acid transporter 1(EAAT1) expression remained stable throughout the period of chronic ocular hypertension. α2-Adrenergic treatment upregulated EAAT1 protein levels (P<0.05). NMDA receptor (GluN1) expression was stimulated by chronic elevation of IOP, and GluN1-positive cells in ganglion cell layer were co-localized with TUNEL staining. Brimonidine administration suppressed GluN1 levels (P<0.05). These results indicate that brimonidine decreased RGC apoptosis, upregulating EAAT1 and downregulating NMDA receptors. We suggest that topical brimonidine treatment may decrease the glutamate excitotoxicity through modulation of glutamate transporter and NMDA receptor in glaucoma. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

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    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.

  18. Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis.

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    Lu, Jun; Wu, Dong-mei; Zheng, Yuan-lin; Hu, Bin; Zhang, Zi-feng

    2010-05-01

    Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, protects brain function against oxidative stress induced by D-galactose (D-gal) (Sigma-Aldrich, St. Louis, MO, USA). Our data showed that PSPC enhanced open-field activity, decreased step-through latency, and improved spatial learning and memory ability in D-gal-treated old mice by decreasing advanced glycation end-products' (AGEs) formation and the AGE receptor (RAGE) expression, and by elevating Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (Sigma-Aldrich) and catalase (CAT) expression and activity. Cleavage of caspase-3 and increased terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL)-positive cells in D-gal-treated old mice were inhibited by PSPC, which might be attributed to its antioxidant property. PSPC also suppressed the activation of c-Jun NH(2)-terminal kinase (JNK) and the release of cytochrome c from mitochondria that counteracted the onset of neuronal apoptosis in D-gal-treated old mice. Furthermore, it was demonstrated that phosphoinositide 3-kinase (PI3K) activation was required for PSPC to promote the neuronal survival accompanied with phosphorylation and activation of Akt and p44/42 mitogen-activated protein kinase (MAPK) by using PI3K inhibitor LY294002 (Cell Signaling Technology, Inc., Beverly, MA, USA), implicating a neuronal survival mechanism. The present results suggest that neuronal survival promoted by PSPC may be a potentially effective method to enhance resistance of neurons to age-related disease.

  19. Sulfotanshinone IIA Sodium Ameliorates Glucose Peritoneal Dialysis Solution-Induced Human Peritoneal Mesothelial Cell Injury via Suppression of ASK1-P38-mediated Oxidative Stress

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    Yao Zhou

    2018-05-01

    Full Text Available Background/Aims: Long-term use of high-glucose peritoneal dialysis solution (PDS induces peritoneal mesothelial cell (PMC injury, peritoneal dysfunction, and peritoneal dialysis (PD failure in patients with end-stage renal disease. How to preserve PMCs in PD is a major challenge for nephrologists worldwide. In this study, we aimed to elucidate the efficacy and mechanisms of sulfotanshinone IIA sodium (Tan IIa in ameliorating high-glucose PDS-induced human PMC injury. Methods: The human PMC line HMrSV5 was incubated with 4.25% PDS in vitro to mimic the high-glucose conditions in PD. Cellular viability was measured by Cell Counting Kit 8. Generation of superoxide and reactive oxygen species (ROS was assessed using a Total ROS/Superoxide Detection Kit. Oxidative modification of protein was evaluated by OxyBlot Protein Oxidation Detection Kit. TUNEL (dT-mediated dUTP nick end labeling assay and DAPI (4,6-diamidino-2-phenylindole staining were used to evaluate apoptosis. Western blot analysis was performed to evaluate the efficacy and mechanisms of Tan IIa. Results: Tan IIa protected PMCs against PDS-induced injury as evidenced by alleviating changes in morphology and loss of cell viability. Consistent with their antioxidant properties, N-acetyl-L-cysteine (NAC and Tan IIa suppressed superoxide and ROS production, protein oxidation, and apoptosis elicited by PDS. Apoptosis signal-regulating kinase 1 (ASK1-p38 signaling was activated by PDS. Both Tan IIa and NAC suppressed ASK1 and p38 phosphorylation elicited by PDS. Moreover, genetic downregulation of ASK1 ameliorated cell injury and inhibited the phosphorylation of p38 and activation of caspase 3. Conclusion: Tan IIa protects PMCs against PDS-induced oxidative injury through suppression of ASK1-p38 signaling.

  20. The 1-Tosylpentan-3-one Protects against 6-Hydroxydopamine-Induced Neurotoxicity

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    Chien-Jen Kao

    2017-05-01

    Full Text Available Previous studies have demonstrated that the marine compound austrasulfone, isolated from the soft coral Cladiella australis, exerts a neuroprotective effect. The intermediate product in the synthesis of austrasulfone, dihydroaustrasulfone alcohol, attenuates several inflammatory responses. The present study uses in vitro and in vivo methods to investigate the neuroprotective effect of dihydroaustrasulfone alcohol-modified 1-tosylpentan-3-one (1T3O. Results from in vitro experiments show that 1T3O effectively inhibits 6-hydroxydopamine-induced (6-OHDA-induced activation of both p38 mitogen-activated protein kinase (MAPK and caspase-3 in SH-SY5Y cells; and enhances nuclear factor erythroid 2–related factor 2 (Nrf2 and heme oxygenase-1 (HO-1 expression via phosphoinositide 3-kinase (PI3K/protein kinase B (Akt signaling. Hoechst staining and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining results reveal that 1T3O significantly inhibits 6-OHDA-induced apoptosis. In addition, the addition of an Akt or HO-1 inhibitor decreases the protective effect of 1T3O. Thus, we hypothesize that the anti-apoptotic activity of 1T3O in neuronal cells is mediated through the regulation of the Akt and HO-1 signaling pathways. In vivo experiments show that 1T3O can reverse 6-OHDA-induced reduction in locomotor behavior ability in zebrafish larvae, and inhibit 6-OHDA-induced tumor necrosis factor-alpha (TNF-α increase at the same time. According to our in vitro and in vivo results, we consider that 1T3O exerts its anti-apoptotic activities at SH-SY5Y cells after 6-OHDA challenges, probably via the regulation of anti-oxidative signaling pathways. Therefore, this compound may be a promising therapeutic agent for neurodegenerations.

  1. Effect of different wavelengths of light on the antioxidant and immunity status of juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress

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    Choe, Jong Ryeol; Shin, Yoon Sub; Choi, Ji Yong; Kim, Tae Hwan; Jung, Min-Min; Choi, Cheol Young

    2017-12-01

    We investigated the effect of light wavelengths on antioxidant and immunity parameters in juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress (25 and 30°C). We exposed the fish to light emitting diodes (LEDs) emitting green (520 nm) and red light (630 nm) of 0.25 and 0.5 W/m2 intensity, and measured the activity, and mRNA and protein expression levels of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. We also determined the levels of plasma hydrogen peroxide (H2O2), melatonin, and lysozyme. Furthermore, the mRNA and protein levels of caspase-3 were measured and terminal transferase dUTP nick end labeling (TUNEL) assays were performed. We observed that mRNA expression and activities of antioxidant enzymes and plasma H2O2 levels were significantly higher after exposure to high temperatures. However, increases in these parameters were significantly lower after exposure to green LED light. The plasma melatonin and lysozyme levels were significantly lower in the different groups after exposure to high temperatures; however, in groups exposed to green LED light, their levels were significantly higher than those in the control group. The expression pattern of caspase-3 mRNA was similar to that of H2O2. The TUNEL assay showed that apoptosis was markedly higher at higher water temperatures than that at 20°C. These results indicate that high water temperatures induce oxidative stress and decrease the immunity in juvenile rock bream but green LED light inhibits the rise in oxidative stress and combats the decrease in immunity and should, thus, be useful in the culture of rock bream.

  2. 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.

  3. PEG-b-PCL polymeric nano-micelle inhibits vascular angiogenesis by activating p53-dependent apoptosis in zebrafish.

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    Zhou, Tian; Dong, Qinglei; Shen, Yang; Wu, Wei; Wu, Haide; Luo, Xianglin; Liao, Xiaoling; Wang, Guixue

    Micro/nanoparticles could cause adverse effects on cardiovascular system and increase the risk for cardiovascular disease-related events. Nanoparticles prepared from poly(ethylene glycol) (PEG)- b -poly( ε -caprolactone) (PCL), namely PEG- b -PCL, a widely studied biodegradable copolymer, are promising carriers for the drug delivery systems. However, it is unknown whether polymeric PEG- b -PCL nano-micelles give rise to potential complications of the cardiovascular system. Zebrafish were used as an in vivo model to evaluate the effects of PEG- b -PCL nano-micelle on cardiovascular development. The results showed that PEG- b -PCL nano-micelle caused embryo mortality as well as embryonic and larval malformations in a dose-dependent manner. To determine PEG- b -PCL nano-micelle effects on embryonic angiogenesis, a critical process in zebrafish cardiovascular development, growth of intersegmental vessels (ISVs) and caudal vessels (CVs) in flk1-GFP transgenic zebrafish embryos using fluorescent stereomicroscopy were examined. The expression of fetal liver kinase 1 (flk1), an angiogenic factor, by real-time quantitative polymerase chain reaction (qPCR) and in situ whole-mount hybridization were also analyzed. PEG- b -PCL nano-micelle decreased growth of ISVs and CVs, as well as reduced flk1 expression in a concentration-dependent manner. Parallel to the inhibitory effects on angiogenesis, PEG- b -PCL nano-micelle exposure upregulated p53 pro-apoptotic pathway and induced cellular apoptosis in angiogenic regions by qPCR and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay. This study further showed that inhibiting p53 activity, either by pharmacological inhibitor or RNA interference, could abrogate the apoptosis and angiogenic defects caused by PEG- b -PCL nano-micelles, indicating that PEG- b -PCL nano-micelle inhibits angiogenesis by activating p53-mediated apoptosis. This study indicates that polymeric PEG- b -PCL nano-micelle could

  4. 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.

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

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    Ju-Ha Kim

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

  6. Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio

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    Azimahtol Hawariah LP

    2007-04-01

    Full Text Available Abstract Background Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines. Results Zerumbone significantly showed an antiproliferative activity upon HepG2 cells with an IC50 of 3.45 ± 0.026 μg/ml. Zerumbone was also found to inhibit the proliferation of non-malignant Chang Liver and MDBK cell lines. However the IC50 obtained was higher compared to the IC50 for HepG2 cells (> 10 μg/ml. The extent of DNA fragmentation was evaluated by the Tdt-mediated dUTP nick end labelling assay which showed that, zerumbone significantly increased apoptosis in HepG2 cells in a time-course manner. In detail, the apoptotic process triggered by zerumbone involved the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression. The changes that occurred in the levels of this antagonistic proteins Bax/Bcl-2, was independent of p53 since zerumbone did not affect the levels of p53 although this protein exists in a functional form. Western blotting analysis for Bax protein was further confirmed qualitatively with an immunoassay that showed the distribution of Bax protein in zerumbone-treated cells. Conclusion Therefore, zerumbone was found to induce the apoptotic process in HepG2 cells through the up and down regulation of Bax/Bcl-2 protein independently of functional p53 activity.

  7. Abnormal Sensory Protein Expression and Urothelial Dysfunction in Ketamine-Related Cystitis in Humans

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    Yao Chou Tsai

    2016-09-01

    Full Text Available Purpose The aim of this study was to analyze patterns of sensory protein expression and urothelial dysfunction in ketamine-related cystitis (KC in humans. Methods Biopsies of bladder mucosa were performed in 29 KC patients during cystoscopy. Then specimens were analyzed for tryptase, zonula occludens-1 (ZO-1, E-cadherin, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL with immunofluorescence staining and quantification. In addition, 10 healthy control bladder specimens were analyzed and compared with the KC specimens. Another 16 whole bladder specimens obtained from partial cystectomy were also analyzed for the muscarinic receptors M2 and M3, endothelial nitric oxide synthase (eNOS, inducible nitric oxide synthase (iNOS, β-3 adrenergic receptors (β3-ARs, and the P2X3 receptor by western blotting. In addition, 3 normal control bladder specimens were analyzed and compared with the KC specimens. Results The KC bladder mucosa revealed significantly less expression of ZO-1 and E-cadherin, and greater expression of TUNEL and tryptase activity than the control samples. The expression of M3 and β3-AR in the KC specimens was significantly greater than in the controls. The expression of iNOS, eNOS, M2, and P2X3 was not significantly different between the KC and control specimens. Conclusions The bladder tissue of KC patients revealed significant urothelial dysfunction, which was associated with mast-cell mediated inflammation, increased urothelial cell apoptosis, and increased expression of the M3 and β3-AR.

  8. Human lactoferrin stimulates skin keratinocyte function and wound re-epithelialization.

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    Tang, L; Wu, J J; Ma, Q; Cui, T; Andreopoulos, F M; Gil, J; Valdes, J; Davis, S C; Li, J

    2010-07-01

    Human lactoferrin (hLF), a member of the transferrin family, is known for its antimicrobial and anti-inflammatory effects. Recent studies on various nonskin cell lines indicate that hLF may have a stimulatory effect on cell proliferation. To study the potential role of hLF in wound re-epithelialization. The effects of hLF on cell growth, migration, attachment and survival were assessed, with a rice-derived recombinant hLF (holo-rhLF), using proliferation analysis, scratch migration assay, calcein-AM/propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method, respectively. The mechanisms of hLF on cell proliferation and migration were explored using specific pathway inhibitors. The involvement of lactoferrin receptor low-density lipoprotein receptor-related protein 1 (LRP1) was examined with RNA interference technique. An in vivo swine second-degree burn wound model was also used to assess wound re-epithelialization. Studies revealed that holo-rhLF significantly stimulated keratinocyte proliferation which could be blocked by mitogen-activated protein kinase (MAPK) kinase 1 inhibitor. Holo-rhLF also showed strong promoting effects on keratinocyte migration, which could be blocked by either inhibition of the MAPK, Src and Rho/ROCK pathways, or downregulation of the LRP1 receptor. With cells under starving or 12-O-tetradecanoylphorbol-13-acetate exposure, the addition of holo-rhLF was found greatly to increase cell viability and inhibit cell apoptosis. Additionally, holo-rhLF significantly increased the rate of wound re-epithelialization in swine second-degree burn wounds. Our studies demonstrate the direct effects of holo-rhLF on wound re-epithelialization including the enhancement of keratinocyte proliferation and migration as well as the protection of cells from apoptosis. The data strongly indicate its potential therapeutic applications in wound healing.

  9. N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes.

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    Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

    2013-03-01

    Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg(-1)). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.

  10. Biological effects of blocking blue and other visible light on the mouse retina.

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    Narimatsu, Toshio; Ozawa, Yoko; Miyake, Seiji; Kubota, Shunsuke; Yuki, Kenya; Nagai, Norihiro; Tsubota, Kazuo

    2014-08-01

    To elucidate the biological effects of blocking fluorescent light on the retina using specific blocking materials. Seven- to 8-week-old BALB/c mice were divided into three groups and placed in one of the three boxes: one blocked ultraviolet and violet wavelengths of light (violet blockade), one blocked ultraviolet, violet, blue and some other visible wavelengths (blue-plus blockade), and one allowed most visible light to pass through (control). They were then exposed to a white fluorescent lamp for 1 h at 5.65E-05 mW/cm(2) /s. After treatment, the electroretinogram, retinal outer nuclear layer thickness and retinal outer segment length were measured. In addition, retinal apoptotic cells were quantified by TdT-mediated dUTP nick-end labelling assay and c-Fos messenger RNA, and protein levels were measured by real-time reverse-transcription polymerase chain reaction and immunoblot analyses, respectively. The blue-plus blockade group retained a significantly better electroretinogram response following light exposure than the control or violet blockade groups. The blue-plus blockade group also exhibited greater outer nuclear layer thickness and greater outer-segment length, and fewer apoptotic cells after light exposure than the other groups. The c-Fos messenger RNA and protein levels were substantially reduced in the blue-plus blockade group and reduced to a lesser extent in the violet blockade group. The blockade of blue plus additional visible wavelengths of light was most effective in protecting the retina from light-induced damage. The blockade of violet light alone was also effective in reducing intracellular molecular responses, but these effects were not sufficient for attenuating retinal degeneration. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

  11. Prognostic value of p53, c-ErbB2 and tunel data in upper urothelial carcinoma associated with Balkan nephropathy

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    Savin Marina

    2014-01-01

    Full Text Available A characteristic tumor suppressor protein 53 (p53 mutational profile of genotoxic action of aristolochic acid was identified in the upper urothelial carcinoma (UUTT associated with Balkan nephropathy (BEN. In the present study, we examined the prognostic value of tissue-based molecular markers in overall-survival (OS risk after surgical treatment of UUTT, adjusted for gender, age and urological characteristics in 32 patients with BEN. Immunohistochemical examination of p53, the proliferation cell nuclear antigen (PCNA, the human epidermal growth factor receptor 2 (c-ErbB2; also known as HER-2/neu proto-oncogene and the in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay for apoptosis detection were used to examine serial tumor sections. The median OS-time was 60 months for UUTT operation; the mortality rate (18.7% was related to (new disease (reoccurrence or invasion in 12-216 months. High-grade (p=0.029, TUNEL>0.36%+ cells (p=0.010, and c-ErbB2+ cells (p=0.014 can define the risk of tumor invasion. Patients with Balkan nephropathy that develop UUTT at a stage greater than pT1 (with apoptosis TUNEL+ cells >0.36% and p53+ cells greater than 10% were at high risk of poor-OS after the tumor surgery (h(x=6.35; p=0.045. The obtained data present evidence for p53, cErbB2 and apoptosis deregulation, as a result of environmental toxin action. This is the first report of molecular biomarker linkage with OS for BEN-associated UUTT.

  12. Sirtuin 1 participates in the process of age-related retinal degeneration.

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    Zeng, Ying; Yang, Ke

    The process of aging involves retinal cell damage that leads to visual dysfunction. Sirtuin (Sirt) 1 can prevent oxidative stress, DNA damage, and apoptosis. In the present study, we measured the expression of Sirt1 as a functional regulator in the retina during the aging process. The visual function and Sirt1 expression in young (1 month) and old (19 months) Sprague-Dawley (SD) rats. Electroretinogram (ERG) and real-time polymerase chain reaction (PCR) or Western blotting were performed. Resveratrol, an activator of Sirt1, was orally administered to SD rats at a dose of 5 mg/kg/day for 19 months. The expression of Sirt1, brain-derived neurotrophic factor (BDNF), and tropomyosin receptor kinase B (TrkB) was evaluated in the retinas of mice that did and did not receive resveratrol treatment. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. With decreasing b-wave amplitude, the expression level of Sirt1 was significantly reduced in aged retinas compared to that in young retinas. After 19 months of treatment with resveratrol, the Sirt1 expression level and b-wave amplitude increased. In old rats treated with resveratrol, the expression levels of BDNF and TrkB were up-regulated. Compared to young retinas, the aged retinas exhibited higher apoptosis, but resveratrol delayed this process. Our data demonstrated a reduction of Sirt1 expression during the aging process of the retina, but enhancing Sirt1 expression reversed the degeneration of the retina. These results suggested that increasing Sirt1 expression may protect retinal neurons and visual function via regulating neurotrophin and its receptor. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic.

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    An, Songhie; Nam, Kihoon; Choi, Sunghyun; Bai, Cheng Z; Lee, Yan; Park, Jong-Sang

    2013-01-01

    Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model. The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG) by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4). Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of .40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, 4',6-Diamidino-2-phenylindole (DAPI) TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining. The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier's high transfection efficiency (35%-40%) in glioma cells and the selective apoptosis-inducing activity of apoptin in tumor cells.

  14. The Protective Effect of Spinal Cord Stimulation Postconditioning Against Spinal Cord Ischemia/Reperfusion Injury in Rabbits.

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    Li, Huixian; Dong, Xiuhua; Jin, Mu; Cheng, Weiping

    2018-01-18

    Delayed paraplegia due to spinal cord ischemia/reperfusion injury (IRI) remains one of the most severe complications of thoracoabdominal aneurysm surgery, for which effective prevention and treatment is still lacking. The current study investigates whether spinal cord stimulation (SCS) postconditioning has neuroprotective effects against spinal cord IRI. Ninety-six New Zealand white male rabbits were randomly divided into four groups as follows: a sham group and three experimental groups (C group, 2 Hz group, and 50 Hz group) n = 24/group. Spinal cord ischemia was induced by transient infrarenal aortic balloon occlusion for 28 min, after which rabbits in group C underwent no additional intervention, while rabbits in the other two experimental groups underwent 2 Hz or 50 Hz epidural SCS for 30 min at the onset of reperfusion and then daily until sacrifice. Hind limb neurologic function of rabbits was assessed using Jacob scale. Lumbar spinal cords were harvested immediately after sacrifice for histological examination and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The number of viable α-motor neurons in ventral horn was counted and TUNEL-positive rate of α-motor neurons was calculated. Spinal cord IRI was caused by transient infrarenal aorta occlusion for 28 min. Both 2 Hz and 50 Hz SCS postconditioning had neuroprotective effects, particularly the 2 Hz SCS postconditioning. Comparing to C group and 50 Hz group, rabbits in the 2 Hz group demonstrated better hind limb motor function and a lower rate of TUNEL-positive α-motor neuron after eight hours, one day, three days, and seven days of spinal cord reperfusion. More viable α-motor neurons were preserved after one and three days of spinal cord reperfusion in 2 Hz group rabbits than in C group and 50 Hz group rabbits. SCS postconditioning at 2 Hz protected the spinal cord from IRI. © 2018 International Neuromodulation Society.

  15. Anatase titanium dioxide nanoparticles in mice: evidence for induced structural and functional sperm defects after short-, but not long-, term exposure

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    Michelle A Smith

    2015-04-01

    Full Text Available 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.

  16. Holotransferrin enhances selective anticancer activity of artemisinin against human hepatocellular carcinoma cells.

    Science.gov (United States)

    Deng, Xiao-rong; Liu, Zhao-xia; Liu, Feng; Pan, Lei; Yu, He-ping; Jiang, Jin-ping; Zhang, Jian-jun; Liu, Li; Yu, Jun

    2013-12-01

    Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine artemesia annua L. (the blue-green herb) in the early 1970s, which has been confirmed for effectively treating malaria. Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill cancer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransferrin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling (TUNEL) and colony formation assay, respectively. The results showed that artemisinin at various concentrations significantly inhibited growth, colony formation and cell viability of SMMC-7721 cells (P<0.05), likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P<0.01). Our data suggest that treatment with artemisinin leads to inhibition of viability and proliferation, and apoptosis of SMMC-7721 cells. Furthermore, we observed that holotransferrin significantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

  17. Growth-Inhibitory and Apoptosis-Inducing Effects of Punica granatum L. var. spinosa (Apple Punice) on Fibrosarcoma Cell Lines.

    Science.gov (United States)

    Sineh Sepehr, Koushan; Baradaran, Behzad; Mazandarani, Masoumeh; Yousefi, Bahman; Abdollahpour Alitappeh, Meghdad; Khori, Vahid

    2014-12-01

    Punica granatum L. var. granatum (Pomegranate), an herbaceous plant found in Iran, The aim of this study was to investigate the cytotoxic effects, induction of apoptosis, and the mechanism of cell death of ethanol extract from Punica granatum L. var. spinosa on the mouse fibrosarcoma cell line, WEHI-164. Various parts of the herbs were extracted from fruit using ethanol as the solvent, and the cytotoxicity and cell viability of the ethanolic extract were determined by the MTT assay. To determine whether necrosis or apoptosis is the predominant cause of cell death, cell death detection was performed using the ELISA method. The induction of apoptosis was confirmed using the terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Moreover, a sensitive immunoblotting technique was used to examine the production of Caspase-3 and Bcl2 proteins. Our findings suggested that the ethalonic extract of Punica granatum L. var. spinosa altered cell morphology, decreased cell viability, suppressed cell proliferation and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 229.024μg/ml), when compared to a chemotherapeutic anticancer drug, Toxol (Vesper Pharmaceuticals), with increased nucleosome production from apoptotic cells. Induction of apoptosis by the plant extract was proved by the decrease of pro-Caspase-3 and Bcl2 proteins and quantitatively confirmed by Immunoblotting analysis. The results obtained from the present study have demonstrated the growth-inhibitory effect of Ethanol Extracts from Punica granatum L. var. spinosa, and clearly showed that apoptosis was the major mechanism of in-vitro cell death induced by the extract.

  18. 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.

  19. Seawater immersion aggravates burn-associated lung injury and inflammatory and oxidative-stress responses.

    Science.gov (United States)

    Ma, Jun; Wang, Ying; Wu, Qi; Chen, Xiaowei; Wang, Jiahan; Yang, Lei

    2017-08-01

    With the increasing frequency of marine development activities and local wars at sea, the incidence of scald burns in marine accidents or wars has been increasing yearly. Various studies have indicated that immersion in seawater has a systemic impact on some organs of animals or humans with burn. Thus, for burn/scald injuries after immersion in seawater, it is desirable to study the effects and mechanisms of action on important organs. In the present study, we aimed to investigate the effect of immersion in seawater on lung injury, inflammatory and oxidative-stress responses in scalded rats. The structural damage to lungs was detected by hematoxylin and eosin staining and the results showed that seawater immersion aggravated structural lung injury in scalded rats. The expression of HMGB1 in lung tissues was detected by immunohistochemical analysis and the results showed that seawater immersion increased HMGB1 expression in lung tissues of scalded rats. Apoptosis in lung tissues was detected by terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling (TUNEL) staining and the results showed that seawater immersion increased apoptosis rate in lung tissues of scalded rats. In addition, the expression levels of TNF-α, IL-6, IL-8, SOD, and MDA in serum were analyzed by enzyme-linked immunosorbent assays (ELISAs) and the results showed that seawater immersion induced secretion of proinflammatory factors (TNF-α, IL-6, and IL-8), increased MDA protein level, and suppressed SOD activity in the serum of scalded rats. Furthermore, measurement of plasma volume and pH showed that seawater immersion decreased plasma volume and pH value. Overall, the results indicated that all effects induced by immersion in seawater in scalded rats are more pronounced than those induced by freshwater. In conclusion, seawater immersion may aggravate lung injury and enhance inflammatory and oxidative-stress responses after burn. Copyright © 2017 Elsevier Ltd and ISBI. All rights

  20. Hepatocyte growth factor gene-modified adipose-derived mesenchymal stem cells ameliorate radiation induced liver damage in a rat model.

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    Jiamin Zhang

    Full Text Available Liver damage caused by radiotherapy is associated with a high mortality rate, but no established treatment exists. Adipose-derived mesenchymal stem cells (ADSCs are capable of migration to injured tissue sites, where they aid in the repair of the damage. Hepatocyte growth factor (HGF is critical for damage repair due to its anti-apoptotic, anti-fibrotic and cell regeneration-promoting effects. This study was performed to investigate the therapeutic effects of HGF-overexpressing ADSCs on radiation-induced liver damage (RILD. ADSCs were infected with a lentivirus encoding HGF and HGF-shRNA. Sprague-Dawley (SD rats received 60Gy of irradiation to induce liver injury and were immediately given either saline, ADSCs, ADSCs + HGF or ADSCs + shHGF. Two days after irradiation, a significant reduction in apoptosis was observed in the HGF-overexpressing ADSC group compared with the RILD group, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining. Scanning electron microscopy showed chromatin condensation after irradiation, which was ameliorated in the group that received ADSCs and was reversed in the group that received HGF-overexpressing ADSCs. HGF-overexpressing ADSCs ameliorated radiation- induced liver fibrosis through down regulation of α-SMA and fibronectin. Hepatocyte regeneration was significantly improved in rats treated with ADSCs compared with rats from the RILD group, as assessed by Ki-67 immunohistochemistry. Rats that received HGF-overexpressing ADSCs showed an even greater level of hepatocyte regeneration. HGF-overexpressing ADSCs completely blocked the radiation-induced increase in the enzymes ALT and AST. The effect of mitigating RILD was compromised in the ADSC + shHGF group compared with the ADSC group. Altogether, these results suggest that HGF-overexpressing ADSCs can significantly improve RILD in a rat model, which may serve as a valuable therapeutic alternative.

  1. [Effect of NOR1 gene knockdown on the biological behavior of HeLa cells].

    Science.gov (United States)

    Tan, Yixin; Li, Wenjuan; Yi, Mei; Wang, Wei; Zheng, Pan; Zhang, Haijing; Xiang, Bo; Li, Guiyuan

    2014-08-01

    To explore the effect of the oxidored nitro domain containing protein 1 (NOR1) gene knockdown on the biological behavior of HeLa cells in cervical carcinoma. The recombinant plasmids pSUPER-shNOR1-1, pSUPER-shNOR1-2 and pSUPERscramble, which targeted to NOR1 gene, were constructed by pSUPER.neo+GFP vector, transfected into HeLa cells respectively using Lipofectamine 2000 reagent, and followed by G418 selection. The expression level of NOR1 mRNA and protein were determined by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the growth curve of cell viability. The stable transfectants were treated with H₂O₂ and cell apoptosis was determined by Hoechst 33258 staining and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. The expression levels of Bcl-2, cleaved caspase 9 and poly ADP-ribose polymerase (PARP) were measured by Western blot. NOR1- knockdown HeLa cells were successfully constructed by transfection of pSUPER-shNOR1-1 or pSUPER-shNOR1-2 plasmids into HeLa cells. MTT assay showed that the silence of endogenous NOR1 in HeLa cells could lead to the increase in cell viability and proliferation, and the inhibition of H₂O₂-induced apoptosis compared with the negative control. Western blot showed that the expression level of active caspase 9 and cleaved PARP was inhibited in NOR1-knockdown cells when they were treated with H₂O₂ while the expression level of Bcl-2 protein increased. Silence of endogenous NOR1 facilitates the cell viability and growth of HeLa cells, and attenuates HeLa cells apoptosis induced by H₂O₂, which might be mediated by up-regulation of Bcl-2 level and down-regulation of the cleaved caspase 9 cascade.

  2. Postconditioning with sevoflurane ameliorates spatial learning and memory deficit via attenuating endoplasmic reticulum stress induced neuron apoptosis in a rat model of hemorrhage shock and resuscitation.

    Science.gov (United States)

    Hu, Xianwen; Wang, Jingxian; Zhang, Li; Zhang, Qiquan; Duan, Xiaowen; Zhang, Ye

    2018-06-02

    Hemorrhage shock could initiate endoplasmic reticulum stress (ERS) and then induce neuronal apoptosis. The aim of this study was to investigate whether sevoflurane postconditioning could attenuate brain injury via suppressing apoptosis induced by ERS. Seventy male rats were randomized into five groups: sham, shock, low concentration (sevo1, 1.2%), middle concentration (sevo2, 2.4%) and high concentration (sevo3, 3.6%) of sevoflurane postconditioning. Hemorrhage shock was induced by removing 40% of the total blood volume during an interval of 30 min. 1h after the completion of bleeding, the animals were reinfused with shed blood during the ensuing 30 min. The spatial learning and memory ability of rats were measured by Morris water maze (MWM) test three days after the operation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the hippocampus CA1 region were assessed after the MWM test. The expression of C/EBP-homologousprotein (CHOP) and glucose-regulated protein 78 (GRP78) in the hippocampus were measured at 24h after reperfusion. We found that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% significantly ameliorated the spatial learning and memory ability, decreased the TUNEL-positive cells, and reduced the GRP78 and CHOP expression compared with the shock group. These results suggested that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% could ameliorate spatial learning and memory deficit after hemorrhage shock and resuscitation injury via suppressing apoptosis induced by ERS. Copyright © 2018. Published by Elsevier B.V.

  3. Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model.

    Science.gov (United States)

    Rigo, Federica; De Stefano, Nicola; Navarro-Tableros, Victor; David, Ezio; Rizza, Giorgia; Catalano, Giorgia; Gilbo, Nicholas; Maione, Francesca; Gonella, Federica; Roggio, Dorotea; Martini, Silvia; Patrono, Damiano; Salizzoni, Mauro; Camussi, Giovanni; Romagnoli, Renato

    2018-05-01

    The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV). We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction. During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014). HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

  4. Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury.

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    Bo Young Jeong

    Full Text Available Contrast-induced acute kidney injury (CIAKI is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH oxidases (Noxs are important sources of reactive oxygen species (ROS. Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831. Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38 implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2'-deoxyguanosine (8OHdG and kidney injury molecule-1 (KIM-1, and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI.

  5. Ochratoxin A Inhibits Mouse Embryonic Development by Activating a Mitochondrion-Dependent Apoptotic Signaling Pathway

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    Yan-Der Hsuuw

    2013-01-01

    Full Text Available Ochratoxin A (OTA, a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.

  6. Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling.

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    Sang-Hun Kang

    Full Text Available BACKGROUND: The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML cell line U937. METHODOLOGY/PRINCIPAL FINDINGS: Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose polymerase (PARP in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-x(L and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2, the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP SH2 domain-containing phosphatase (SHP-1, and the addition of sodium pervanadate (a PTP inhibitor prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.

  7. Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae).

    Science.gov (United States)

    Flores-Rentería, Lluvia; Orozco-Arroyo, Gregorio; Cruz-García, Felipe; García-Campusano, Florencia; Alfaro, Isabel; Vázquez-Santana, Sonia

    2013-09-01

    The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described. The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD. PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption. Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.

  8. Glutamine reduces myocardial cell apoptosis in a rat model of sepsis by promoting expression of heat shock protein 90.

    Science.gov (United States)

    Li, Wanxia; Tao, Shaoyu; Wu, Qinghua; Wu, Tao; Tao, Ran; Fan, Jun

    2017-12-01

    Myocardial cell injury and cardiac myocyte apoptosis are associated with sepsis. Glutamine (Gln) has been reported to repair myocardial cell injury. The aim of this study was to explore the role of Gln on cardiac myocytes in a cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Following induction of sepsis in a CLP rat model, viral encoding heat shock protein 90 (Hsp90) gene and Hsp90dsDNA were designed to express and knockdown Hsp90, respectively. Rat cardiac tissues were examined histologically, and apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein, Hsp90, p53 upregulated modulator of apoptosis, and p53 was measured by western blotting and real-time polymerase chain reaction. Caspase-3, caspase-8, and caspase-9 were detected by enzyme-linked immunosorbent assay. Rat cardiac myocyte damage induced by CLP was reduced by Gln treatment and Hsp90 overexpression, and these changes were reversed by Hsp90 knockdown. Bcl-2 expression, Bcl-2-associated X protein, p53, p53 upregulated modulator of apoptosis, caspase-8, caspase-9, and caspase-3 activities were significantly upregulated in the CLP model, which were reduced by Gln treatment and Hsp90 overexpression. Gln reduced apoptosis of cardiac myocytes in a rat model of sepsis, by promoting Hsp90 expression. Further studies are needed to determine the possible therapeutic action of Gln in sepsis in human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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 (TiO2) 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. PMID:25370207

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    Highlights: • Murine NTDs were successfully induced by means of hydroxyurea (HU). • The impairment of dNTP was induced via inhibition of ribonucleotide reductase. • dNTP deficiency induced by HU caused defective DNA synthesis and repair. • Abnormal apoptosis and proliferation induced by HU affected neural tube development. - Abstract: 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

  11. DJ-1 Modulates Nuclear Erythroid 2-Related Factor-2-Mediated Protection in Human Primary Alveolar Type II Cells in Smokers.

    Science.gov (United States)

    Bahmed, Karim; Messier, Elise M; Zhou, Wenbo; Tuder, Rubin M; Freed, Curt R; Chu, Hong Wei; Kelsen, Steven G; Bowler, Russell P; Mason, Robert J; Kosmider, Beata

    2016-09-01

    Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.

  12. Targeting Gene-Viro-Therapy with AFP driving Apoptin gene shows potent antitumor effect in hepatocarcinoma

    Directory of Open Access Journals (Sweden)

    Zhang Kang-Jian

    2012-02-01

    Full Text Available Abstract Background Gene therapy and viral therapy are used for cancer therapy for many years, but the results are less than satisfactory. Our aim was to construct a new recombinant adenovirus which is more efficient to kill hepatocarcinoma cells but more safe to normal cells. Methods By using the Cancer Targeting Gene-Viro-Therapy strategy, Apoptin, a promising cancer therapeutic gene was inserted into the double-regulated oncolytic adenovirus AD55 in which E1A gene was driven by alpha fetoprotein promoter along with a 55 kDa deletion in E1B gene to form AD55-Apoptin. The anti-tumor effects and safety were examined by western blotting, virus yield assay, real time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide assay, Hoechst33342 staining, Fluorescence-activated cell sorting, xenograft tumor model, Immunohistochemical assay, liver function analysis and Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay. Results The recombinant virus AD55-Apoptin has more significant antitumor effect for hepatocelluar carcinoma cell lines (in vitro than that of AD55 and even ONYX-015 but no or little impair on normal cell lines. Furthermore, it also shows an obvious in vivo antitumor effect on the Huh-7 liver carcinoma xenograft in nude mice with bigger beginning tumor volume till about 425 mm3 but has no any damage on the function of liver. The induction of apoptosis is involved in AD55-Apoptin induced antitumor effects. Conclusion The AD55-Apoptin can be a potential anti-hepatoma agent with remarkable antitumor efficacy as well as higher safety in cancer targeting gene-viro-therapy system.

  13. Anti-oxidative and anti-inflammatory effects of cinnamaldehyde on protecting high glucose-induced damage in cultured dorsal root ganglion neurons of rats.

    Science.gov (United States)

    Yang, Dan; Liang, Xiao-Chun; Shi, Yue; Sun, Qing; Liu, Di; Liu, Wei; Zhang, Hong

    2016-01-01

    To examine the mechanism underlying the beneficial role of cinnamaldehyde on oxidative damage and apoptosis in high glucose (HG)-induced dorsal root ganglion (DRG) neurons in vitro. HG-treated DRG neurons were developed as an in vitro model of diabetic neuropathy. The neurons were randomly divided into five groups: the control group, the HG group and the HG groups treated with 25, 50 and 100 nmol/L cinnamaldehyde, respectively. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis rate was evaluated by the in situ TdT-mediated dUTP nick end labeling (TUNEL) assay. The intracellular level of reactive oxygen species (ROS) was measured with flow cytometry. Expression of nuclear factor-kappa B (NF-κB), inhibitor of κB (IκB), phosphorylated IκB (p-IκB), tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and caspase-3 were determined by western blotting and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) were also measured by western blotting. Cinnamaldehyde reduced HG-induced loss of viability, apoptosis and intracellular generation of ROS in the DRG neurons via inhibiting NF-κB activity. The western blot assay results showed that the HG-induced elevated expressions of NF-κB, IκB and p-IκB were remarkably reduced by cinnamaldehyde treatment in a dose-dependent manner (P neurons, but also lowered the elevated IL-6, TNF-α, cyclo-oxygenase and inducible nitric oxide synthase levels, indicating a reduction in inflammatory damage. Cinnamaldehyde protected DRG neurons from the deleterious effects of HG through inactivation of NF-κB pathway but not through activation of Nrf2/HO-1. And thus cinnamaldehyde may have potential application as a treatment for DPN.

  14. 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. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  15. Protective effects of a green tea polyphenol, epigallocatechin-3-gallate, against sevoflurane-induced neuronal apoptosis involve regulation of CREB/BDNF/TrkB and PI3K/Akt/mTOR signalling pathways in neonatal mice.

    Science.gov (United States)

    Ding, Mei-Li; Ma, Hui; Man, Yi-Gang; Lv, Hong-Yan

    2017-12-01

    Epigallocatechin-3-gallate (EGCG), a polyphenol in green tea, is an effective antioxidant and possesses neuroprotective effects. Brain-derived neurotrophic factor (BDNF) and cyclic AMP response element-binding protein (CREB) are crucial for neurogenesis and synaptic plasticity. In this study, we aimed to assess the protective effects of EGCG against sevoflurane-induced neurotoxicity in neonatal mice. Distinct groups of C57BL/6 mice were given EGCG (25, 50, or 75 mg/kg body weight) from postnatal day 3 (P3) to P21 and were subjected to sevoflurane (3%; 6 h) exposure on P7. EGCG significantly inhibited sevoflurane-induced neuroapoptosis as determined by Fluoro-Jade B staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Increased levels of cleaved caspase-3, downregulated Bad and Bax, and significantly enhanced Bcl-2, Bcl-xL, xIAP, c-IAP-1, and survivin expression were observed. EGCG induced activation of the PI3K/Akt pathway as evidenced by increased Akt, phospho-Akt, GSK-3β, phospho-GSK-3β, and mTORc1 levels. Sevoflurane-mediated downregulation of cAMP/CREB and BDNF/TrkB signalling was inhibited by EGCG. Reverse transcription PCR analysis revealed enhanced BDNF and TrkB mRNA levels upon EGCG administration. Improved performance of mice in Morris water maze tests suggested enhanced learning and memory. The study indicates that EGCG was able to effectively inhibit sevoflurane-induced neurodegeneration and improve learning and memory retention of mice via activation of CREB/BDNF/TrkB-PI3K/Akt signalling.

  16. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Wu, Honghai; Gai, Renhua; Wu, Youping [Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); Yang, Bo [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Yang, Xiaochun, E-mail: yangxiaochun@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China)

    2012-06-15

    Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

  17. Toxicity assessment of perfluorooctane sulfonate using acute and subchronic male C57BL/6J mouse models

    International Nuclear Information System (INIS)

    Xing, Jiali; Wang, Gang; Zhao, Jichun; Wang, Eryin; Yin, Boxing; Fang, Dongsheng; Zhao, Jianxin; Zhang, Hao; Chen, Yong Q.; Chen, Wei

    2016-01-01

    Perfluorooctane sulfonate (PFOS) is a principal representative and the final degradation product of several commercially produced perfluorinated compounds. However, PFOS has a high bioaccumulation potential and therefore can exert toxicity on aquatic organisms, animals, and cells. Considering the widespread concern this phenomenon has attracted, we examined the acute and subchronic toxic effects of varying doses of PFOS on adult male C57BL/6 mice. The acute oral LD_5_0 value of PFOS in male C57BL/6J mice was 0.579 g/kg body weight (BW). Exposure to the subchronic oral toxicity of PFOS at 2.5, 5, and 10 mg PFOS/kg BW/day for 30 days disrupted the homeostasis of antioxidative systems, induced hepatocellular apoptosis (as revealed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay), triggered liver injury (as evidenced by the increased serum levels of aspartate aminotransferase, alanine amino transferase, alkaline phosphatase, and gamma-glutamyl transpeptidase and by the altered histology), and ultimately increased the liver size and relative weight of the mice. PFOS treatment caused liver damage but only slightly affected the kidneys and spleen of the mice. This study provided insights into the toxicological effects of PFOS. - Highlights: • The acute and subchronic toxicity of PFOS was systematically investigated. • The acute oral LD_5_0 value for PFOS in C57BL/6J mice was 0.579 g/kg body weight. • PFOS disrupted the homeostasis of antioxidative systems. • PFOS induced hepatocellular apoptosis and triggered liver injury. - PFOS disrupted the homeostasis of antioxidative systems, induced hepatocellular apoptosis, and triggered liver injury.

  18. Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes

    Science.gov (United States)

    Bernerd, Francoise; Sarasin, Alain; Magnaldo, Thierry

    1999-01-01

    Galectin-7 is a β-galactoside binding protein specifically expressed in stratified epithelia and notably in epidermis, but barely detectable in epidermal tumors and absent from squamous carcinoma cell lines. Galectin-7 gene is an early transcriptional target of the tumor suppressor protein P53 [Polyak, K., Xia, Y., Zweier, J., Kinzler, K. & Vogelstein, B. (1997) Nature (London) 389, 300–305]. Because p53 transcriptional activity is increased by genotoxic stresses we have examined the possible effects of ultraviolet radiations (UVB) on galectin-7 expression in epidermal keratinocytes. The amounts of galectin-7 mRNA and protein are increased rapidly after UVB irradiation of epidermal keratinocytes. The increase of galectin-7 is parallel to P53 stabilization. UVB irradiation of skin reconstructed in vitro and of human skin ex vivo demonstrates that galectin-7 overexpression is associated with sunburn/apoptotic keratinocytes. Transfection of a galectin-7 expression vector results in a significant increase in terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive keratinocytes. The present findings demonstrate a keratinocyte-specific protein involved in the UV-induced apoptosis, an essential process in the maintenance of epidermal homeostasis. PMID:10500176

  19. Lidový hudebně-taneční projev jako scénický tvar - folklorní soubory v českých zemích v péči jedné instituce

    Czech Academy of Sciences Publication Activity Database

    Stavělová, Daniela

    2015-01-01

    Roč. 25, č. 4 (2015), s. 292-306 ISSN 0862-8351 Institutional support: RVO:68378076 Keywords : folk dance * folk music * stage * show * institution * folklore ensemble * folklore stylization Subject RIV: AC - Archeology, Anthropology, Ethnology

  20. Effect of limb ischemic preconditioning on myocardial apoptosis-related proteins in ischemia-reperfusion injury

    Science.gov (United States)

    GAO, JIANZHI; ZHAO, LINJING; WANG, YONGLING; TENG, QINGLEI; LIANG, LIDONG; ZHANG, JINYING

    2013-01-01

    The aim of this study was to investigate the effect of limb ischemic preconditioning (LIPC) on myocardial apoptosis in myocardial ischemia-reperfusion injury (MIRI), as well as the regulation of caspase-3 and the B cell lymphoma 2 (Bcl-2) gene in LIPC. A total of 50 rats were divided randomly into 5 groups (n=10). Four rats in each group were drawn out for detection of apoptosis. The sham, MIRI and LIPC groups underwent surgery without additional treatment. In the LY294002 group, LY294002 preconditioning was administered 15 min before reperfusion. In the LY294002+LIPC group, following LIPC, LY294002 was administered 15 min before reperfusion. The relative expression of myocardial Bcl-2 and caspase-3 mRNA and the apoptotic index for each group were determined by reverse transcription-polymerase chain reaction (RT-PCR) and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), respectively. The ultrastructure of the cardiac muscle tissues was observed by election microscopy. Compared with the sham group, the expression of caspase-3 mRNA in the MIRI group significantly increased (P<0.05) and the expression of Bcl-2 mRNA clearly decreased. Compared with the MIRI group, LIPC reduced the expression of caspase-3 and increased the expression of Bcl-2 mRNA (P<0.05). There were no significant differences between the LY294002+LIPC group and the MIRI group. Compared with the sham group, the apoptotic index of myocardial cells in the MIRI group significantly increased (P<0.05). Compared with the MIRI group, LIPC significantly decreased the apoptotic index of myocardial cells (P<0.05) and LY294002 increased the apoptotic index of myocardial cells. Compared with the LIPC group, LY294002+LIPC significantly increased the apoptotic index of myocardial cells (P<0.05). There were no significant differences between the LY294002+LIPC and MIRI groups. In conclusion, LIPC increased the expression of Bcl-2 and decreased caspase-3 mRNA and

  1. Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial-mesenchymal transition-induced angiogenesis.

    Science.gov (United States)

    Li, C; Li, Q; Cai, Y; He, Y; Lan, X; Wang, W; Liu, J; Wang, S; Zhu, G; Fan, J; Zhou, Y; Sun, R

    2016-09-01

    Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck and is associated with a high rate of lymph node metastasis. The initial step in the metastasis and transition of tumors is epithelial-mesenchymal transition (EMT)-induced angiogenesis, which can be mediated by angiopoietin 2 (ANG2), a key regulatory factor in angiogenesis. In the present study, immunohistochemistry and real-time quantitative reverse transcriptase (qRT-PCR) were used to measure the expression of ANG2 in OSCC tissues. Plasmids encoding ANG2 mRNA were used for increased ANG2 expression in the OSCC cell line TCA8113. The short interfering RNA (siRNA)-targeting ANG2 mRNA sequences were used to inhibit ANG2 expression in TCA8113 cells. Subsequently, transwell assays were performed to examine the effects of ANG2 on TCA8113 cell migration and invasion. Furthermore, in vivo assays were performed to assess the effect of ANG2 on tumor growth. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemistry were used to examine cell apoptosis and angiogenesis in tumor tissues, respectively. Finally, western blot analysis was performed to evaluate tumor formation-related proteins in OSCC tissues. We found that protein expression of ANG2 was remarkably upregulated in OSCC tissues. Overexpression of ANG2 increased the migration and invasion of TCA8113 cells by regulating EMT. Further investigations showed that overexpression of ANG2 increased tumor growth in nude mice, and angiogenesis of OSCC tissues increased in the presence of ANG2 overexpression. Overexpression of ANG2 also reduced cell apoptosis in tumor tissue cells. Finally, we found that overexpression of ANG2 resulted in changes in the expression of tumor formation-related proteins including vimentin, E-cadherin, Bim, PUMA, Bcl-2, Bax, Cyclin D1, PCNA and CD31. Our findings show that ANG2 has an important role in the migration and invasion of OSCC. More importantly, further

  2. Overexpression of HIPK2 attenuates spinal cord injury in rats by modulating apoptosis, oxidative stress, and inflammation.

    Science.gov (United States)

    Li, Renbo; Shang, Jingbo; Zhou, Wei; Jiang, Li; Xie, Donghui; Tu, Guanjun

    2018-04-09

    HIPK2 is considered to be a tumor suppressor. It also has been implicated in several functions such as apoptosis and inflammation that are linked to spinal cord injury (SCI). However, whether HIPK2 ameliorates the neurological pain of SCI remains unclear. Here, we investigated the effects of HIPK2 on neurological function, oxidative stress, levels of inflammatory cytokines and expression of Bcl-2/Bax in an SCI model. Firstly, we evaluated the therapeutic effects of HIPK2 on neurological pain in the SCI rat using the Basso, Beattie and Bresnahan scores and H & E staining. Overexpression of HIPK2 significantly elevated the levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), and reduced the mRNA expression of Nogo-A and RhoA in SCI rats. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays showed that overexpression of HIPK2 significantly reduced the number of apoptotic cells. Overexpression of HIPK2 also decreased expression of Bax and Caspase-3 and elevated expression of Bcl-2 in the SCI model, indicating that HIPK2 exhibited its protective activity by inhibiting SCI-induced apoptosis. Then, we measured the serum concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). We also determined the mRNA and protein levels of nuclear factor-κB p65 unit, tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β. HIPK2 overexpression reduced oxidative stress and the levels of inflammatory cytokines compared with SCI control animals. Additionally, acetylation of HIPK2 was reduced in SCI rats. Overexpression of HIPK2 could enhance autophagy by elevating the expression of Beclin-1 and LC3-II while autophagy is regarded as a beneficial regulator to improve spinal cord injury. Together, overexpression of HIPK2 improved contusive SCI induced pain by modulating oxidative stress, Bcl‑2 and Bax signaling, and

  3. Differential responses to high- and low-dose ultraviolet-B stress in tobacco Bright Yellow-2 cells

    Science.gov (United States)

    Takahashi, Shinya; Kojo, Kei H.; Kutsuna, Natsumaro; Endo, Masaki; Toki, Seiichi; Isoda, Hiroko; Hasezawa, Seiichiro

    2015-01-01

    Ultraviolet (UV)-B irradiation leads to DNA damage, cell cycle arrest, growth inhibition, and cell death. To evaluate the UV-B stress–induced changes in plant cells, we developed a model system based on tobacco Bright Yellow-2 (BY-2) cells. Both low-dose UV-B (low UV-B: 740 J m−2) and high-dose UV-B (high UV-B: 2960 J m−2) inhibited cell proliferation and induced cell death; these effects were more pronounced at high UV-B. Flow cytometry showed cell cycle arrest within 1 day after UV-B irradiation; neither low- nor high-UV-B–irradiated cells entered mitosis within 12 h. Cell cycle progression was gradually restored in low-UV-B–irradiated cells but not in high-UV-B–irradiated cells. UV-A irradiation, which activates cyclobutane pyrimidine dimer (CPD) photolyase, reduced inhibition of cell proliferation by low but not high UV-B and suppressed high-UV-B–induced cell death. UV-B induced CPD formation in a dose-dependent manner. The amounts of CPDs decreased gradually within 3 days in low-UV-B–irradiated cells, but remained elevated after 3 days in high-UV-B–irradiated cells. Low UV-B slightly increased the number of DNA single-strand breaks detected by the comet assay at 1 day after irradiation, and then decreased at 2 and 3 days after irradiation. High UV-B increased DNA fragmentation detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay 1 and 3 days after irradiation. Caffeine, an inhibitor of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) checkpoint kinases, reduced the rate of cell death in high-UV-B–irradiated cells. Our data suggest that low-UV-B–induced CPDs and/or DNA strand-breaks inhibit DNA replication and proliferation of BY-2 cells, whereas larger contents of high-UV-B–induced CPDs and/or DNA strand-breaks lead to cell death. PMID:25954287

  4. The effects of erdosteine, N-acetylcysteine, and vitamin E on nicotine-induced apoptosis of pulmonary cells

    International Nuclear Information System (INIS)

    Demiralay, Rezan; Guersan, Nesrin; Erdem, Havva

    2006-01-01

    This study was conducted to investigate the frequency of apoptosis in the pulmonary epithelial cells of rats after intratraperitoneal nicotine injection, in order to examine the role of inflammatory markers [myeloperoxidase (MPO) and tumor necrosis factor-alpha (TNF-α)] in nicotine-induced lung damage, and to determine the protective effects of three known antioxidant agents [N-acetylcysteine (NAC), erdosteine, and vitamin E] on the lung toxicity of nicotine in the lungs. Female Wistar rats were divided into seven groups, each composed of nine rats: two negative control groups, two 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 injected intraperitoneally 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. Lung tissue samples were stained with hematoxylin-eosin (H and E) for histopathological assessments. The apoptosis level in the lung bronchiolar and alveolar epithelium was determined by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. Cytoplasmic TNF-α in the bronchiolar and alveolar epithelial cells and the lung MPO activity were evaluated immunohistochemically. The protective effect of vitamin E on lung histology was stronger than that of erdosteine or NAC. Treatment with erdosteine, NAC, and vitamin E significantly reduced the rate of nicotine-induced pulmonary epithelial cell apoptosis, and there were no significant differences in apoptosis among the three antioxidants groups. Erdosteine, NAC, and vitamin E significantly reduced the increases in TNF-α staining and lung MPO activity. The effects of erdosteine on the increases in the local TNF-α level and lung MPO activity were weaker than that of NAC or vitamin E. This findings suggest that erdosteine and NAC can be as effective as vitamin

  5. Echinophora platyloba DC (Apiaceae crude extract induces apoptosis in human prostate adenocarcinoma cells (PC 3

    Directory of Open Access Journals (Sweden)

    Fatemeh Zare Shahneh

    2014-10-01

    Full Text Available Background: Prostate cancer is the second leading malignancy worldwide and the second prominent cause of cancer-related deaths among men. Therefore, there is a serious necessity for finding advanced alternative therapeutic measures against this lethal malignancy. In this article, we report the cytotoxicity and the mechanism of cell death of the methanolic extract prepared from Echinophora platyloba DC plant against human prostate adenocarcinoma PC 3 cell line and Human Umbilical Vein Endothelial Cells HUVEC cell line. Methods: Cytotoxicity and viability of the methanolic extract were assessed by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and dye exclusion assay. Cell death enzyme-linked immunosorbent assay (ELISA was employed to quantify the nucleosome production resulting 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 and DNA fragmentation gel electrophoresis. Results: E. platyloba could decrease cell viability in malignant cells in a dose- and time-dependent manner. The IC50 values against PC 3 were determined as 236.136 ± 12.4, 143.400 ± 7.2, and 69.383 ± 1.29 μg/ml after 24, 36, and 48 h, respectively, but there was no significant activity in HUVEC normal cell (IC50 > 800 μg/ml. Morphological characterizations and DNA laddering assay showed that the methanolic extract treated cells displayed marked apoptotic characteristics such as nuclear fragmentation, appearance of apoptotic bodies, and DNA laddering fragment. Increase in an early apoptotic population was observed in a dose-dependent manner. PC 3 cell death elicited by the extract was found to be apoptotic in nature based a clear indication of TUNEL assay and gel electrophoresis DNA fragmentation, which is a hallmark of apoptosis

  6. Niclosamide suppresses hepatoma cell proliferation via the Wnt pathway

    Directory of Open Access Journals (Sweden)

    Tomizawa M

    2013-11-01

    Full Text Available Minoru Tomizawa,1 Fuminobu Shinozaki,2 Yasufumi Motoyoshi,3 Takao Sugiyama,4 Shigenori Yamamoto,5 Makoto Sueishi,4 Takanobu Yoshida6 1Department of Gastroenterology, 2Department of Radiology, 3Department of Neurology, 4Department of Rheumatology, 5Department of Pediatrics, 6Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Chiba, Japan Background: The Wnt pathway plays an important role in hepatocarcinogenesis. We analyzed the association of the Wnt pathway with the proliferation of hepatoma cells using Wnt3a and niclosamide, a drug used to treat tapeworm infection. Methods: We performed an MTS assay to determine whether Wnt3a stimulated proliferation of Huh-6 and Hep3B human hepatoma cell lines after 72 hours of incubation with Wnt3a in serum-free medium. The cells were subjected to hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL after 48 hours of incubation. RNA was isolated 48 hours after addition of Wnt3a or niclosamide, and cyclin D1 expression levels were analyzed by real-time quantitative polymerase chain reaction. The promoter activity of T-cell factor was analyzed by luciferase assay 48 hours after transfection of TOPflash. Western blot analysis was performed with antibodies against β-catenin, dishevelled 2, and cyclin D1. Results: Cell proliferation increased with Wnt3a. Niclosamide suppressed proliferation with or without Wnt3a. Hematoxylin and eosin and TUNEL staining suggested that apoptosis occurred in cells with niclosamide. Cyclin D1 was upregulated in the presence of Wnt3a and downregulated with addition of niclosamide. The promoter activity of T-cell factor increased with Wnt3a, whereas T-cell factor promoter activity decreased with niclosamide. Western blot analysis showed that Wnt3a upregulated β-catenin, dishevelled 2, and cyclin D1, while niclosamide downregulated them. Conclusion: Niclosamide is a potential

  7. In vivo prediction of anti-tumor effect of 3-bromopyruvate in hepatocellular carcinoma using Tc-99m labeled annexin-v imaging

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    Kim, Won; Yoon, Jung Hwan; Kim, Chung Yang [Seoul National University College of Medicine, Seoul (Korea, Republic of); Cheon, Gi Jeoog; Lee, Tae Sup; Woo, Kwang Sun; Chung, Wee Sup [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2005-07-01

    We have recently demonstrated that hypoxia stimulates hepatocellular carcinoma (HCC) cell growth through hexokinase II induction, and its inhibition induces apoptotic cell death through activating mitochondrial apoptotic signaling cascades. In this study, we were apt to evaluate the antitumoral effect of 3-bromopyruvate (3-BP) on in vivo model of HCC by apoptotic imaging using Tc-99m labeled annexin V. In vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells, a mouse HCC cell line, and 3-BP (0, 5, 10 mg/kg) was subsequently administered intraperitoneally. Tc-99m-HYNIC-annexin V (185 KBq) was injected via tail vein at one and three days after the 3-BP treatment, planar scan was acquired at a hour after the injection using gamma camera. The anti-tumor effect was evaluated by measuring tumor volumes and quantification of apoptotic cells using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Tumor volume was significantly reduced in mice treated with 3-BP in a dose-dependent manner (mean tumor volume 1.07 vs. 0.58 vs. 0.39 cm{sup 3} in 3-BP 0, 5, 10 mg/kg, respectively: p=0.047). The percentage of TUNEL staining-positive cells was significantly increased in 3-BP-treated mice (0.53 vs. 1.40 vs. 1.84% in 3-BP 0, 5, 10 mg/kg, respectively; p=0.018). On Tc-99m-HYNIC annexin V imaging, tumor-to-background uptake ratio (UR) was 1.92 at one day and 4.23 at three days after 3-BP treatment of 5 mg/kg (non-treated tumor showed UR of 2.93). Apoptosis-inducing anti-tumor effect of 3-BP was able to be demonstrated in in vivo model of HCC by apoptotic in vivo imaging using Tc-99m-HYNIC annexin V.

  8. In vivo prediction of anti-tumor effect of 3-bromopyruvate in hepatocellular carcinoma using Tc-99m labeled annexin-v imaging

    International Nuclear Information System (INIS)

    Kim, Won; Yoon, Jung Hwan; Kim, Chung Yang; Cheon, Gi Jeoog; Lee, Tae Sup; Woo, Kwang Sun; Chung, Wee Sup

    2005-01-01

    We have recently demonstrated that hypoxia stimulates hepatocellular carcinoma (HCC) cell growth through hexokinase II induction, and its inhibition induces apoptotic cell death through activating mitochondrial apoptotic signaling cascades. In this study, we were apt to evaluate the antitumoral effect of 3-bromopyruvate (3-BP) on in vivo model of HCC by apoptotic imaging using Tc-99m labeled annexin V. In vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells, a mouse HCC cell line, and 3-BP (0, 5, 10 mg/kg) was subsequently administered intraperitoneally. Tc-99m-HYNIC-annexin V (185 KBq) was injected via tail vein at one and three days after the 3-BP treatment, planar scan was acquired at a hour after the injection using gamma camera. The anti-tumor effect was evaluated by measuring tumor volumes and quantification of apoptotic cells using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Tumor volume was significantly reduced in mice treated with 3-BP in a dose-dependent manner (mean tumor volume 1.07 vs. 0.58 vs. 0.39 cm 3 in 3-BP 0, 5, 10 mg/kg, respectively: p=0.047). The percentage of TUNEL staining-positive cells was significantly increased in 3-BP-treated mice (0.53 vs. 1.40 vs. 1.84% in 3-BP 0, 5, 10 mg/kg, respectively; p=0.018). On Tc-99m-HYNIC annexin V imaging, tumor-to-background uptake ratio (UR) was 1.92 at one day and 4.23 at three days after 3-BP treatment of 5 mg/kg (non-treated tumor showed UR of 2.93). Apoptosis-inducing anti-tumor effect of 3-BP was able to be demonstrated in in vivo model of HCC by apoptotic in vivo imaging using Tc-99m-HYNIC annexin V

  9. Protective effect of DA-9401 in finasteride-induced apoptosis in rat testis: inositol requiring kinase 1 and c-Jun N-terminal kinase pathway

    Directory of Open Access Journals (Sweden)

    Soni KK

    2017-10-01

    Full Text Available Kiran Kumar Soni,1,* Yu Seob Shin,1,* Bo Ram Choi,1 Keshab Kumar Karna,1 Hye Kyung Kim,2 Sung Won Lee,3 Chul Young Kim,4 Jong Kwan Park1 1Department of Urology, Chonbuk National University and Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute and Clinical Trial Center of Medical Device of Chonbuk National University, Jeonju, 2College of Pharmacy, Kyungsung University, Busan, 3Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University Medical School, Seoul, 4College of Pharmacy, Hangyang University, Ansan, Republic of Korea *These authors contributed equally to this work Abstract: Finasteride is used to treat male pattern baldness and benign prostatic hyperplasia. This study investigated the toxicity of finasteride and recovery by DA-9401 using Sprague Dawley (SD rats. Forty adult male SD rats were assigned to four groups: control (CTR, finasteride 1 mg/kg/day (F, finasteride 1 mg/kg + DA-9401 100 mg/kg/day (F + DA 100 and finasteride 1 mg/kg + DA-9401 200 mg/kg/day (F + DA 200. Treatments were by oral delivery once daily for 90 consecutive days. The gross anatomical parameters assessed included: genital organ weight; vas deferens sperm count and sperm motility; testosterone, dihydrotestosterone (DHT and malondialdehyde levels; and histological and terminal deoxynucleotidyl transferase enzyme mediated dUTP nick-end labeling (TUNEL staining of testis for spermatogenic cell density, Johnsen’s score and apoptosis. Testicular tissue was also used for evaluating endoplasmic reticulum (ER stress and apoptotic proteins. Epididymis weight, seminal vesicle weight, prostate weight, penile weight and vas deferens sperm motility showed significant differences between the F group and the CTR, F + DA 100 and F + DA 200 groups. There was no significant change in the testosterone level. DHT level decreased significantly in the F group compared with the CTR

  10. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling

    Science.gov (United States)

    ITO, TAKUJI; BAI, TAO; TANAKA, TETSUJI; YOSHIDA, KENJI; UEYAMA, TAKASHI; MIYAJIMA, MASAYASU; NEGISHI, TAKAYUKI; KAWASAKI, TAKAHIKO; TAKAMATSU, HYOTA; KIKUTANI, HITOSHI; KUMANOGOH, ATSUSHI; YUKAWA, KAZUNORI

    2015-01-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild-type (WT) mice. Administration of β-estradiol to infant Sema4D-deficient (Sema4D−/−) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β-estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin-B1, was examined as well as the level of apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin-B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase-3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five-week-old Sema4D−/− mice compared with WT mice. The addition of recombinant Sema4D to Sema4D−/− vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis-inducing activity of Sema4D. The experimental reduction of

  11. Dual Inhibition of Topoisomerase II and Tyrosine Kinases by the Novel Bis-Fluoroquinolone Chalcone-Like Derivative HMNE3 in Human Pancreatic Cancer Cells.

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    Yong-Chao Ma

    Full Text Available Both tyrosine kinase and topoisomerase II (TopII are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT dUTP nick-end labeling (TUNEL assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK detection kit using a horseradish peroxidase (HRP-conjugated phosphotyrosine (pY20 antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P<0.05, and this effect was accompanied by a decrease in tyrosine kinase activity. HMNE3 potentially inhibited tyrosine kinase activity in vitro with an IC50 value of 0.64±0.34 μmol/L in Capan-1 cells and 3.1±0.86 μmol/L in Panc-1 cells. The activity of c-Src was significantly inhibited by HMNE3 in a dose

  12. Effects of AVX-470, an Oral, Locally Acting Anti-Tumour Necrosis Factor Antibody, on Tissue Biomarkers in Patients with Active Ulcerative Colitis.

    Science.gov (United States)

    Hartman, Deborah S; Tracey, Daniel E; Lemos, Brenda R; Erlich, Emma C; Burton, Randall E; Keane, David M; Patel, Rutvij; Kim, Skaison; Bhol, Kailash C; Harris, M Scott; Fox, Barbara S

    2016-06-01

    AVX-470 is an orally administered, bovine-derived, anti-tumour necrosis factor (TNF) antibody with local activity in the gastrointestinal tract. In the first-in-human clinical trial of AVX-470 in active ulcerative colitis, we evaluated inflammatory biomarkers in colon tissue as measures of disease activity and early response to treatment. Thirty-six patients received active drug (AVX-470 at 0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Colon biopsy samples were collected from 5 regions of colon at baseline and week 4. Tissue inflammatory biomarkers were evaluated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), epithelial cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and bovine immunoglobulin by immunohistochemistry and mass spectrometry. Endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity [UCEIS]) at colonoscopy was assessed in each colonic region by a central reader. Bovine immunoglobulin was observed in mucosal tissue before and after dosing in lamina propria and submucosal layers of biopsy tissue. Baseline levels of TNF, myeloperoxidase (MPO), CD68 and interleukin (IL)-1β and, to a lesser extent, IL-6 mRNA were 2- to 3-fold higher in distal vs proximal colon tissue, corresponding to the 2- to 3-fold differences in baseline severities of endoscopic scores. Reductions of >10-fold in TNF and, to lesser extents, in MPO and epithelial cell apoptosis were observed in proximal and distal colon biopsies after 4 weeks of AVX-470 3.5g/day treatment. Reductions in TNF scores were correlated with changes in MPO and CD3 immunohistochemistry scores. These results are consistent with anti-TNF activity of orally administered AVX-470 in colon mucosal tissue in ulcerative colitis patients and demonstrate the utility of tissue biomarkers in assessing disease and treatment response in early clinical studies. This trial was registered with Clinicaltrials.gov as study NCT

  13. Soya-saponins induce intestinal inflammation and barrier dysfunction in juvenile turbot (Scophthalmus maximus).

    Science.gov (United States)

    Gu, Min; Jia, Qian; Zhang, Zhiyu; Bai, Nan; Xu, Xiaojie; Xu, Bingying

    2018-06-01

    Soybean meal-induced enteritis (SBMIE) is a well-described condition in the distal intestine (DI) of several cultured fish species, but the exact cause is still unclear. The work on Atlantic salmon and zebrafish suggested soya-saponins, as heat-stable anti-nutritional factors in soybean meal, are the major causal agents. However, this conclusion was not supported by the research on some other fish, such as gilthead sea bream and European sea bass. Our previous work proved that soybean could induce SBMIE on turbot and the present work aimed to investigate whether soya-saponins alone could cause SBMIE and the effects of soya-saponins on the intestinal barrier function in juvenile turbot. Turbots with initial weight 11.4 ± 0.02 g were fed one of four fishmeal-based diets containing graded levels of soya-saponins (0, 2.5, 7.5, 15 g kg -1 ) for 8 weeks. At the end of the trial, all fish were weighed and plasma was obtained for diamine oxidase (DAO) activity and d-lactate level analysis and DI was sampled for histological evaluation and quantification of antioxidant parameters and inflammatory marker genes. The activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and intestinal glutathione level were selected to evaluated intestinal antioxidant system. The distal intestinal epithelial cell (IEC) proliferation and apoptosis were investigated by proliferating cell nuclear antigen (PCNA) labelling and TdT-mediated dUTP nick end labeling (TUNEL), respectively. The results showed that soya-saponins caused significantly dose-dependent decrease in the growth performance and nutrient utilization (p soya-saponins. Significantly dose-dependent increases in severity of the inflammation concomitant with up-regulated expression of il-1β, il-8, and tnf-α, increased IEC proliferation and apoptosis, and decreases in selected antioxidant parameters were detected (p soya-saponins (p soya-saponins induced enteritis and compromised

  14. Phytoestrogens/insoluble fibers and colonic estrogen receptor β: Randomized, double-blind, placebo-controlled study

    Science.gov (United States)

    Principi, Mariabeatrice; Di Leo, Alfredo; Pricci, Maria; Scavo, Maria Principia; Guido, Raffaella; Tanzi, Sabina; Piscitelli, Domenico; Pisani, Antonio; Ierardi, Enzo; Comelli, Maria Cristina; Barone, Michele

    2013-01-01

    AIM: To assess the safety and effect of the supplementation of a patented blend of dietary phytoestrogens and insoluble fibers on estrogen receptor (ER)-β and biological parameters in sporadic colonic adenomas. METHODS: A randomized, double-blind placebo-controlled trial was performed. Patients scheduled to undergo surveillance colonoscopy for previous sporadic colonic adenomas were identified, and 60 eligible patients were randomized to placebo or active dietary intervention (ADI) twice a day, for 60 d before surveillance colonoscopy. ADI was a mixture of 175 mg milk thistle extract, 20 mg secoisolariciresinol and 750 mg oat fiber extract. ER-β and ER-α expression, apoptosis and proliferation (Ki-67 LI) were assessed in colon samples. RESULTS: No adverse event related to ADI was recorded. ADI administration showed a significant increases in ER-β protein (0.822 ± 0.08 vs 0.768 ± 0.10, P = 0.04) and a general trend to an increase in ER-β LI (39.222 ± 2.69 vs 37.708 ± 5.31, P = 0.06), ER-β/ER-α LI ratio (6.564 ± 10.04 vs 2.437 ± 1.53, P = 0.06), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (35.592 ± 14.97 vs 31.541 ± 11.54, P = 0.07) and Ki-67 (53.923 ± 20.91 vs 44.833 ± 10.38, P = 0.07) approximating statistical significance. A significant increase of ER-β protein (0.805 ± 0.13 vs 0.773 ± 0.13, P = 0.04), mRNA (2.278 ± 1.19 vs 1.105 ± 1.07, P < 0.02) and LI (47.533 ± 15.47 vs 34.875 ± 16.67, P < 0.05) and a decrease of ER-α protein (0.423 ± 0.06 vs 0.532 ± 0.11, P < 0.02) as well as a trend to increase of ER-β/ER-α protein in ADI vs placebo group were observed in patients without polyps (1.734 ± 0.20 vs 1.571 ± 0.42, P = 0.07). CONCLUSION: The role of ER-β on the control of apoptosis, and its amenability to dietary intervention, are supported in our study. PMID:23885143

  15. High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells.

    Science.gov (United States)

    Fu, J; Tay, S S W; Ling, E A; Dheen, S T

    2006-05-01

    Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs. Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2'-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically. High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells. The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the

  16. Influence of Schistosoma japonicum programmed cell death protein 10 on the growth and development of schistosomula.

    Science.gov (United States)

    Gao, Yan Ru; Huang, Wen Ling; Tang, Chun Lian; Liu, Rong; Zhao, Qin Ping; Ming, Zhen Ping; Dong, Hui Fen

    2018-01-18

    Schistosomiasis caused by Schistosoma japonicum is among the most serious endemic zoonoses in China. To study interactions between schistosomula, the pre-adult juvenile stage, and hosts, it is important to study the functions of key genes involved in schistosomula growth and development. Programmed cell death protein 10 (pcdp10) is an important apoptosis-related gene with various biological functions. This study described the molecular characterization of S. japonicum PCDP10 (SjPCDP10) and evaluated its functions in schistosomula. Real-time quantitative polymerase chain reaction (qPCR) and western blot were used to detect Sjpcdp10 mRNA and protein levels, respectively, at different developmental stages. Immunolocalization was performed to determine SjPCDP10 expression in the parasite. RNA interference (RNAi) experiments were used to assess gene functions associated with SjPCDP10 in schistosomula growth and development. Real-time qPCR revealed that Sjpcdp10 was expressed during all investigated developmental stages and upregulated during schistosomula growth and development. Histochemical localization showed that SjPCDP10 was mainly distributed in the teguments of schistosomula in all investigated stages and part of the parenchymal area of 14-, 18-, and 21-day-old schistosomula. Following Sjpcdp10 knockdown by RNAi, the lengths, widths, areas, and volumes of schistosomula were significantly lower than those in the control group. Scanning electron microscopy showed that the body surfaces of schistosomula subjected to RNAi were seriously damaged, with few tegumental spines and sensory papillae. Transmission electron microscopy indicated that the teguments of Sjpcdp10-knockdown schistosomula were incomplete, the number of layers was reduced, and the thickness decreased significantly as compared with those in the control group. Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end labelling results showed that the rate of apoptosis in Sjpcdp10-knockdown

  17. Quercetin-mediated synthesis of graphene oxide–silver nanoparticle nanocomposites: a suitable alternative nanotherapy for neuroblastoma

    Directory of Open Access Journals (Sweden)

    Yuan YG

    2017-08-01

    Full Text Available Yu-Guo Yuan,1 Yan-Hong Wang,1 Hui-Hui Xing,1 Sangiliyandi Gurunathan2 1Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, People’s Republic of China; 2Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea Background: Graphene and graphene-related materials have gained substantial interest from both academia and industry for the development of unique nanomaterials for biomedical applications. Graphene oxide (GO and silver nanoparticles (AgNPs are a valuable platform for the development of nanocomposites, permitting the combination of nanomaterials with different physical and chemical properties to generate novel materials with improved and effective functionalities in a single platform. Therefore, this study was conducted to synthesize a graphene oxide–silver nanoparticle (GO-AgNPs nanocomposite using the biomolecule quercetin and evaluate the potential cytotoxicity and mechanism of GO-AgNPs in human neuroblastoma cancer cells (SH-SY5Y.Methods: The synthesized GO-AgNPs were characterized using various analytical techniques. The potential toxicities of GO-AgNPs were evaluated using a series of biochemical and cellular assays. The expression of apoptotic and anti-apoptotic genes was measured by quantitative real-time reverse transcription polymerase chain reaction. Further, apoptosis was confirmed by caspase-9/3 activity and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and GO-AgNPs-induced autophagy was also confirmed by transmission electron microscopy.Results: The prepared GO-AgNPs exhibited significantly higher cytotoxicity toward SH-SY5Y cells than GO. GO-AgNPs induced significant cytotoxicity in SH-SY5Y cells by the loss of cell viability, inhibition of cell proliferation, increased leakage of lactate dehydrogenase, decreased level of

  18. Circulating Long Noncoding RNA HOTAIR is an Essential Mediator of Acute Myocardial Infarction

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    Lu Gao

    2017-12-01

    Full Text Available Background/Aims: Acute myocardial infarction (AMI is one of the leading causes of death in the world. However, specific diagnostic biomarkers have not been fully determined, and candidate regulatory targets for AMI have not been identified to date. Long noncoding RNAs (lncRNAs are a class of RNA molecules that have diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases, particularly AMI, is still in its infancy. HOX antisense intergenic RNA (HOTAIR, a 2.2 kb lncRNA, was initially described as a modulator of HOX gene expression. Recent studies have illustrated the important role of HOTAIR in cancer progression, but few studies have reported its function in cardiac disease, including AMI. In the current study, we aimed to detect the expression of HOTAIR during AMI and to explore its function in hypoxia-induced cardiomyocyte injury in neonatal cardiomyocytes. Methods: In 50 consecutively enrolled AMI patients, we examined the serum expression levels of HOTAIR and analysed its correlation with cardiac troponin I (cTnI expression. Another 50 age- and sex-matched subjects served as healthy controls. Next, the HOTAIR expression was detected in the serum from C57BL/6J mice subjected to coronary artery ligation and in neonatal rat cardiomyocytes induced by hypoxia. Cultured cardiomyocytes apoptosis were measured by terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL staining. A search for miRNAs that had complementary base paring with HOTAIR was performed utilizing an online software program, and the interaction between miR-1 and HOTAIR was examined using a luciferase reporter assay. Results: Our study revealed that HOTAIR expression was significantly decreased in the serum of AMI patients compared with that of the healthy controls. Similarly, we observed that HOTAIR was downregulated in the serum of mice subjected to

  19. An experimental study on the antitumor effect of 131I-17-AAG in vitro and in vivo

    International Nuclear Information System (INIS)

    Tu Wenyong; Liu Lu; Chen Daozhen; Huang Ying; Yin Weidong

    2009-01-01

    The objective of this study was to observe the antitumor effect of 131 I-17-allylamino-17-demethoxygeldanamycin ( 131 I-17-AAG) in vitro/in vivo and explore its antitumor mechanism with a view to its potential therapeutic application. 131 I-17-AAG was prepared by the reaction of 17-AAG with Na [ 131 I] in the presence of hydrogen peroxide. The effects of 131 17-AAG on cell growth inhibition and cell cycle distribution in vitro were studied in BEL-7402 cells lines. Following BEL-7402 tumor implantation by subcutaneous xenografts into nude mice, the reagents were injected through the tail vein, and the tumor volume was measured and analyzed. At the end of the experiment, tumor specimens were processed for histopathological analysis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis. The expression change of Akt2 was tested by Western-blot analysis. Methyl-thiazolyl-tetrazolium assay showed inhibition rates of 27.7±5.3%, 57.3±4.3%, and 63.7±3.1%, in Na 131 I group, 17-AAG group, and 131 I-17-AAG group, respectively. The inhibition rate in the 131 I-17-AAG group differed significantly between Na 131 I group and 17-AAG group (F=229.49, P 131 I group, 17-AAG group, and 131 I-17-AAG group, respectively. Following infusion for 32 days, the tumor volumes in the 131 I-17-AAG group were significantly smaller than those in the DMSO group (F=24.18, P 131 I group (F=20.68, P 131 I-17-AAG inhibited the proliferation of tumor cells and induced apoptosis. The expression of Akt2 in 131 I-17-AAG was significantly lower than that in the DMSO group or 131 I group. 131 I-17-AAG can effectively inhibit the growth of BEL-7402 tumor cells in vitro and in vivo. 131 I-17-AAG is a promising agent for the treatment of BEL-7402 cell tumor. (author)

  20. Saponin 6 derived from Anemone taipaiensis induces U87 human malignant glioblastoma cell apoptosis via regulation of Fas and Bcl‑2 family proteins.

    Science.gov (United States)

    Ji, Chen-Chen; Tang, Hai-Feng; Hu, Yi-Yang; Zhang, Yun; Zheng, Min-Hua; Qin, Hong-Yan; Li, San-Zhong; Wang, Xiao-Yang; Fei, Zhou; Cheng, Guang

    2016-07-01

    Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor, and is associated with a poor prognosis. Saponin 6, derived from Anemone taipaiensis, exerts potent cytotoxic effects against the human hepatocellular carcinoma HepG2 cell line and the human promyelocytic leukemia HL‑60 cell line; however, the effects of saponin 6 on glioblastoma remain unknown. The present study aimed to evaluate the effects of saponin 6 on human U87 malignant glioblastoma (U87 MG) cells. The current study revealed that saponin 6 induced U87 MG cell death in a dose‑ and time‑dependent manner, with a half maximal inhibitory concentration (IC50) value of 2.83 µM after treatment for 48 h. However, saponin 6 was needed to be used at a lesser potency in HT‑22 cells, with an IC50 value of 6.24 µM. Cell apoptosis was assessed by flow cytometry using Annexin V‑fluorescein isothiocyanate/propidium iodide double staining. DNA fragmentation and alterations in nuclear morphology were examined by terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling and transmission electron microscopy, respectively. The present study demonstrated that treatment with saponin 6 induced cell apoptosis in U87 MG cells, and resulted in DNA fragmentation and nuclear morphological alterations typical of apoptosis. In addition, flow cytometric analysis revealed that saponin 6 was able to induce cell cycle arrest. The present study also demonstrated that saponin 6‑induced apoptosis of U87 MG cells was attributed to increases in the protein expression levels of Fas, Fas ligand, and cleaved caspase‑3, ‑8 and ‑9, and decreases in the levels of B‑cell lymphoma 2. The current study indicated that saponin 6 may exhibit selective cytotoxicity toward U87 MG cells by activating apoptosis via the extrinsic and intrinsic pathways. Therefore, saponin 6 derived from A. taipaiensis may possess therapeutic potential for the treatment of GBM.

  1. Protective action of tetramethylpyrazine on the medulla oblongata in rats with chronic hypoxia.

    Science.gov (United States)

    Ding, Yan; Hou, Xuefei; Chen, Li; Li, Hui; Tang, Yuhong; Zhou, Hua; Zhao, Shu; Zheng, Yu

    2013-01-01

    Tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Lingusticum Wallichii Frantchat (Chuan Xiong), plays an important role in neuroprotection. However, the protective effect of TMP on the medulla oblongata, the most important region of the brain for cardiovascular and respiratory control, during chronic hypoxia remains unclear. In this study, we examined the neuroprotective effect of TMP on the medulla oblongata after chronic hypoxic injury in rats. Male Sprague-Dawley rats were randomly divided into four groups: control group, TMP group, chronic hypoxia group, and chronic hypoxia+TMP group. Rats were exposed to hypoxia (10% (v/v) O₂) or normoxia for 6 h daily for 14 days. TMP (80 mg/kg) or vehicle (saline) was injected intraperitoneally 30 min before experimentation. Loss of neurons in the pre-Bötzinger complex, the nucleus ambiguus, the nucleus tractus solitarius, the hypoglossal nucleus and the facial nucleus were evaluated by Nissl staining. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured, and apoptosis was monitored using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. The level of Bcl-2 mRNA and Bax mRNA was quantitatively measured by RT-PCR analysis. TMP protected Nissl bodies of neurons from injury in all nuclei observed, and reduced the loss of neurons in the nucleus ambiguus, the nucleus tractus solitarius, and the hypoglossal nucleus in rats subjected to chronic hypoxia. TMP upregulated SOD activity and inhibited the increase in MDA content in the medulla oblongata of hypoxic rats. In addition, TMP decreased the rate of apoptosis index (the percentage of apoptotic cells against the total number of cells) in all medullary structures examined, excepting the nucleus ambiguus and inhibited the decrease in Bcl-2 mRNA levels in the medulla oblongata following hypoxia. Our findings indicate that TMP may protect the medullary structures that are involved in

  2. Arctigenin Treatment Protects against Brain Damage through an Anti-Inflammatory and Anti-Apoptotic Mechanism after Needle Insertion

    Science.gov (United States)

    Song, Jie; Li, Na; Xia, Yang; Gao, Zhong; Zou, Sa-feng; Kong, Liang; Yao, Ying-Jia; Jiao, Ya-Nan; Yan, Yu-Hui; Li, Shao-Heng; Tao, Zhen-Yu; Lian, Guan; Yang, Jing-Xian; Kang, Ting-Guo

    2016-01-01

    Convection enhanced delivery (CED) infuses drugs directly into brain tissue. Needle insertion is required and results in a stab wound injury (SWI). Subsequent secondary injury involves the release of inflammatory and apoptotic cytokines, which have dramatic consequences on the integrity of damaged tissue, leading to the evolution of a pericontusional-damaged area minutes to days after in the initial injury. The present study investigated the capacity for arctigenin (ARC) to prevent secondary brain injury and the determination of the underlying mechanism of action in a mouse model of SWI that mimics the process of CED. After CED, mice received a gavage of ARC from 30 min to 14 days. Neurological severity scores (NSS) and wound closure degree were assessed after the injury. Histological analysis and immunocytochemistry were used to evaluated the extent of brain damage and neuroinflammation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to detect universal apoptosis. Enzyme-linked immunosorbent assays (ELISA) was used to test the inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10) and lactate dehydrogenase (LDH) content. Gene levels of inflammation (TNF-α, IL-6, and IL-10) and apoptosis (Caspase-3, Bax and Bcl-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these, we analyzed ARC’s efficacy and mechanism of action. Results: ARC treatment improved neurological function by reducing brain water content and hematoma and accelerating wound closure relative to untreated mice. ARC treatment reduced the levels of TNF-α and IL-6 and the number of allograft inflammatory factor (IBA)- and myeloperoxidase (MPO)-positive cells and increased the levels of IL-10. ARC-treated mice had fewer TUNEL+ apoptotic neurons and activated caspase-3-positive neurons surrounding the lesion than controls, indicating increased neuronal survival. Conclusions: ARC treatment confers

  3. Arctigenin Confers Neuroprotection Against Mechanical Trauma Injury in Human Neuroblastoma SH-SY5Y Cells by Regulating miRNA-16 and miRNA-199a Expression to Alleviate Inflammation.

    Science.gov (United States)

    Song, Jie; Li, Na; Xia, Yang; Gao, Zhong; Zou, Sa-Feng; Yan, Yu-Hui; Li, Shao-Heng; Wang, Yue; Meng, Ya-Kun; Yang, Jing-Xian; Kang, Ting-Guo

    2016-09-01

    Mechanical trauma injury is a severe insult to neural cells. Subsequent secondary injury involves the release of inflammatory factors that have dramatic consequences for undamaged cells, leading to normal cell death after the initial injury. The present study investigated the capacity for arctigenin (ARC) to prevent secondary effects and evaluated the mechanism underlying the action of microRNA (miRNA)-199a and miRNA-16 in a mechanical trauma injury (MTI) model using SH-SY5Y cells in vitro. SH-SY5Y cells are often applied to in vitro models of neuronal function and differentiation. Recently, miRNAs have been demonstrated to play a crucial role in NF-κB and cholinergic signaling, which can regulate inflammation. The cell model was established by scratch-induced injury of human SH-SY5Y cells, which mimics the characteristics of MTI. A cell counting kit-8 (CCK-8), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunocytochemistry were used to measure cell viability. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inflammatory cytokine and cholinesterase (CHE) content. The lactate dehydrogenase (LDH) content was measured to assess the degree of cell injury. The mRNA levels were measured by RT-PCR to analyze ARC's mechanism of action. miRNA inhibitors and mimics were used to inhibit and strengthen the expression of miRNAs. Protein expression was detected by western blotting analysis. ARC treatment reduced the TNF-α and IL-6 levels as well as the number of TUNEL+ apoptotic SH-SY5Y cells surrounding the scratch and increased the IL-10 level compared to the controls. ARC attenuated the increase of the cell damage degree and LDH content induced by scratching, indicating increased cell survival. Mechanistic studies showed that ARC upregulated the miRNA-16 and miRNA-199a levels to reduce upstream protein (IKKα and IKKβ) expression and inhibit NF-κB signaling pathway activity; moreover, the increased miRNA-199a suppresses

  4. Rhizome extracts of Curcuma zedoaria Rosc induce caspase dependant apoptosis via generation of reactive oxygen species in filarial parasite Setaria digitata in vitro.

    Science.gov (United States)

    Senathilake, K S; Karunanayake, E H; Samarakoon, S R; Tennekoon, K H; de Silva, E D

    2016-08-01

    Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 μg/mL for hexane extract, 191.14 μg/mL for chloroform extract) and adult parasites (IC50 = 77.07 μg/mL for hexane extract, 259.87 μg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST

  5. Protective effect of active perfusion in porcine models of acute myocardial ischemia

    Science.gov (United States)

    Feng, Zanxiang; Mao, Zhifu; Dong, Shengjun; Liu, Baohui

    2016-01-01

    Mortality rates associated with off-pump coronary artery bypass (CAB) are relatively high, as the majority of patients requiring CAB are at a high risk for cardiac events. The present study aimed to establish porcine models of acute myocardial ischemia, and evaluate the protective role of shunt and active perfusion. A total of 30 pigs were randomly assigned to five groups, as follows: i) Sham (control); ii) A1 (shunt; stenosis rate, 55%); iii) A2 (shunt; stenosis rate, 75%); iv) B1 (active perfusion; stenosis rate, 55%); and v) B2 (active perfusion; stenosis rate, 75%) groups. Aortic pressure (P0), left anterior descending coronary pressure (P1), and coronary effective perfusion pressure (P1/P0) were measured. The expression levels of tumor necrosis factor-α (TNF-α), cardiac troponin (cTnI), creatine kinase-myocardial band (CK-MB), interleukin (IL)-6, IL-10, B-cell lymphoma 2 (Bcl-2), and caspase-3 were detected using enzyme-linked immunosorbent assay or western blotting. The myocardial apoptosis rate was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Ischemia models with stenosis rates of 55 and 75% were successfully constructed following suturing of the descending artery. Compared with the control, the 55 and 75% stenosis groups demonstrated significantly decreased P1/P0, increased expression levels of TNF-α, cTnI, CK-MB, IL-6, IL-10 and caspase-3, an increased rate of myocardial apoptosis, and a decreased expression level of anti-apoptotic protein, Bcl-2. At 30 min following successful establishment of the model (ST segment elevation to 1 mm), group B demonstrated significantly increased P1/P0, decreased expression levels of TNF-α, cTnI, CK-MB, IL-6, IL-10 and caspase-3, a decreased rate of myocardial apoptosis, and an increased expression level of anti-apoptotic protein, Bcl-2. Furthermore, the current study indicated that active perfusion was more efficacious in maintaining myocardial perfusion and alleviating

  6. Curcumin Attenuates Hepatotoxicity Induced by Zinc Oxide Nanoparticles in Rats

    Directory of Open Access Journals (Sweden)

    Layasadat Khorsandi

    2016-06-01

    Full Text Available Background: Zinc oxide nanoparticles (NZnO are increasingly used in modern life. Most metal nanoparticles have adverse effects on the liver. Aims: To explore the protective action of curcumin (Cur against hepatotoxicity induced by NZnO in rats. Study Design: Animal experimentation. Methods: Control group animals received normal saline, while the Cur group animals were treated with 200 mg/kg of Cur orally for 21 days. NZnO-intoxicated rats received 50 mg/kg of NZnO for 14 days by gavage method. In the NZnO+Cur group, rats were pretreated with Cur for 7 days before NZnO administration. Plasma activities of Alanine aminotransferase (ALT, aspartate aminotransferase (AST and alkaline phosphatase (ALP were measured as biomarkers of hepatotoxicity. Hepatic levels of malondialdehyde (MDA and superoxide dismutase (SOD and glutathione peroxidase (GPx activities were measured for detection of oxidative stress in liver tissue. Histological changes and apoptosis in liver tissue were studied by using Hematoxylin-eosin staining and the transferase dUTP nick end labeling (TUNEL method. Results: NZnO induced a significant increase in plasma AST (2.8-fold, ALT (2.7-fold and ALP (1.97-fold activity in comparison to the control group (p<0.01. NZnO increased MDA content and reduced SOD and GPx activities. NZnO caused liver damage including centrilobular necrosis and microvesicular steatosis. The percentage of apoptosis in hepatocytes was increased in NZnO-treated rats (p<0.01. Pre-treatment of Cur significantly reduced lipid peroxidation (39%, increased SOD (156% and GPx (26% activities, and attenuated ALT (47%, AST (41% and ALP (30% activities. Pre-treatment with Cur also decreased the histology changes and apoptotic index of hepatocytes (p<0.05. Conclusion: These findings indicate that Cur effectively protects against NZnO-induced hepatotoxicity in rats. However, future studies are required to propose Cur as a potential protective agent against hepatotoxicity

  7. Silencing of B7-H4 suppresses the tumorigenicity of the MGC-803 human gastric cancer cell line and promotes cell apoptosis via the mitochondrial signaling pathway.

    Science.gov (United States)

    Zhou, Donghui; Zhou, Yong; Li, Chao; Yang, Lina

    2018-04-01

    B7-H4 is a transmembrane protein which is a member of the B7 superfamily. It is overexpressed in various types of cancer, including gastric cancer. However, the effects of B7-H4 on the tumorigenicity of gastric cancer and the underlying mechanisms have not yet been fully explored. Thus, the aim of this study was to examine the effects of B7-H4 on the tumorigenicity of gastric cancer cells and to elucidate the underlying mechanisms. For this purpose, B7-H4 expression in gastric cancer tissues was detected by immunohistochemical staining. The effects of B7-H4 on the biological behavior of the MGC-803 human gastric cancer cell line were examined by Cell Counting kit-8 (CCK-8) assay, cell cycle analysis, wound healing assay, Annexin V/propidium iodide staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Moreover, the expression levels of apoptotic markers, such as cleaved caspase‑3, cleaved caspase‑9, Bcl-2 and Bax were examined by western blot analysis. Immunohistochemical staining revealed that a high expression of B7-H4 was found in about 41.8% of tissues obtained from patients with gastric cancer. Comparative analysis revealed that B7-H4 expression significantly correlated with lymph node metastasis and the TNM stage. The results of CCK-8 assay, cell cycle analysis, wound healing assay, Annexin V/propidium iodide staining assay and TUNEL assay all demonstrated that the silencing of B7-H4 by small interfering RNA decreased cell proliferation, suppressed cell motility, and induced cell cycle arrest and the apoptosis of MGC-803 human gastric cancer cells. Furthermore, the results of western blot analysis indicated that the downregulation of B7-H4 induced the apoptosis of the MGC-803 cells via the mitochondrial signaling pathway through the activation of caspase‑3 and caspase‑9, and by altering the Bax/Bcl-2 ratio in a manner that favored apoptosis. Based on the findings on human gastric cancer cell line MGC-803, the

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

    Science.gov (United States)

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

    2017-01-01

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

  9. Evaluation of biodistribution and anti-tumor effect of a dimeric RGD peptide-paclitaxel conjugate in mice with breast cancer

    International Nuclear Information System (INIS)

    Cao, Qizhen; Li, Zi-Bo; Chen, Kai; Wu, Zhanhong; He, Lina; Chen, Xiaoyuan; Neamati, Nouri

    2008-01-01

    Targeting drugs to receptors involved in tumor angiogenesis has been demonstrated as a novel and promising approach to improve cancer treatment. In this study, we evaluated the anti-tumor efficacy of a dimeric RGD peptide-paclitaxel conjugate (RGD2-PTX) in an orthotopic MDA-MB-435 breast cancer model. To assess the effect of conjugation and the presence of drug moiety on the MDA-MB-435 tumor and normal tissue uptake, the biodistribution of 3 H-RGD2-PTX was compared with that of 3 H-PTX. The treatment effect of RGD2-PTX and RGD2+PTX was measured by tumor size, 18 F-FDG/PET, 18 F-FLT/PET, and postmortem histopathology. By comparing the biodistribution of 3 H-RGD2-PTX and 3 H-PTX, we found that 3 H-RGD2-PTX had higher initial tumor exposure dose and prolonged tumor retention than 3 H-PTX. Metronomic low-dose treatment of breast cancer indicated that RGD2-PTX is significantly more effective than PTX+RGD2 combination and solvent control. Although in vivo 18 F-FLT/PET imaging and ex vivo Ki67 staining indicated little effect of the PTX-based drug on cell proliferation, 18 F-FDG/PET imaging showed significantly reduced tumor metabolism in the RGD2-PTX-treated mice versus those treated with RGD2+PTX and solvent control. Terminal uridine deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining also showed that RGD2-PTX treatment also had significantly higher cell apoptosis ratio than the other two groups. Moreover, the microvessel density was significantly reduced after RGD2-PTX treatment as determined by CD31 staining. Our results demonstrate that integrin-targeted delivery of paclitaxel allows preferential cytotoxicity to integrin-expressing tumor cells and tumor vasculature. The targeted delivery strategies developed in this study may also be applied to other chemotherapeutics for selective tumor killing. (orig.)

  10. Dietary nitrate protects submandibular gland from hyposalivation in ovariectomized rats via suppressing cell apoptosis.

    Science.gov (United States)

    Xu, Yipu; Pang, Baoxing; Hu, Liang; Feng, Xiaoyu; Hu, Lei; Wang, Jingsong; Zhang, Chunmei; Wang, Songlin

    2018-02-26

    Xerostomia, a major oral symptom of menopause, is a subjective feeling of dry mouth associated with oral pain and difficulties in deglutition and speech, which significantly reduces patient's quality of life. Dietary nitrate, which can be converted to nitric oxide, has multiple physiological functions in the body, including antioxidant activity and vasodilatation; however, its protective effect against xerostomia remains poorly understood. The present study aimed to evaluate the effects of dietary nitrate on estrogen deficiency-induced xerostomia. We established an ovariectomized (OVX) rat model, which included five groups: sham-operated, OVX, OVX + 0.4 mM nitrate, OVX + 2 mM nitrate, and OVX + 4 mM nitrate (n = 6). After ovariectomy, animals in the nitrate treatment groups received appropriate amounts of sodium nitrate dissolved in distilled water for 3 months. The results showed that nitrate treatment reduced body weight and water intake, and increased serum nitrate and nitrite levels. Furthermore, nitrate uptake increased saliva secretion as evidenced by saliva flow rates and aquaporin 5 expression, and alleviated histological lesions as evidenced by reduction of the fibrotic area and cell atrophy in the salivary glands. Although protective effects of nitrate against estrogen deficiency-induced xerostomia were observed at all doses, treatment with 2 mM nitrate was more effective than that with 0.4 mM and 4 mM nitrate. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 expression analyses showed that nitrate also protected cells from apoptosis, possibly through upregulation of Cu-Zn superoxide dismutase (Cu-Zn SOD) known to inhibit oxidative stress-related apoptosis. Our findings indicate that nitrate could improve functional activity of the salivary glands in OVX rats by suppressing apoptosis and upregulating Cu-Zn SOD expression, suggesting that dietary nitrate may potentially prevent hyposalivation in menopausal

  11. Effect of the addition of two superoxide dismutase analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation.

    Science.gov (United States)

    Santiani, Alexei; Acosta, Alexei Santiani; Evangelista, Shirley; Vargas, Shirley Evangelista; Valdivia, Martha; Cuya, Martha Valdivia; Risopatrón, Jennie; González, Jennie Risopatrón; Sánchez, Raúl; Gutiérrez, Raúl Sánchez

    2013-03-15

    The main objective was to study the effects, on sperm function, of the addition of two superoxide dismutase (SOD) analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. Twelve alpaca semen samples were collected using an artificial vagina and then diluted at a 1:3 ratio in an extender based on skim milk, egg yolk, and fructose. Each semen sample was divided into three equal parts to form the following groups: control, Tempo (1 mM), and Tempol (1 mM). Groups were cooled to 5 °C in 90 minutes (-1 °C in 3 minutes); when samples reached approximately 10 °C, SOD analogues were added to the respective groups. At 5 °C, ethylene glycol (final concentration, 0.1 M) was added to each group. After 30 minutes at 5 °C, samples were loaded in 0.25 mL plastic straws, placed in liquid nitrogen vapor for 15 minutes, and then plunged. Percentages of sperm motility, functional sperm membrane integrity, and viable sperm with intact acrosomes were evaluated before and after freeze-thaw using visual analysis, the hypoosmotic swelling test, and the double-stain trypan blue/giemsa technique, respectively. The Terminal deoxymucleotidyl transferase dUTP Nick End Labeling assay was performed for evaluation of sperm DNA fragmentation of frozen-thawed sperm. Sperm motility was higher (P < 0.05) in the Tempol and Tempo groups than in the control group (mean, 22.1%, 19.7%, and 11.2%, respectively), with similar results for functional sperm membrane integrity. Additionally, DNA fragmentation was lower (P < 0.05) in the Tempol group (16.7%) than in the control group (38.8%). Viable sperm with intact acrosomes were not affected by the use of SOD analogues. There was a negative correlation (r = -0.58) between DNA fragmentation of alpaca sperm and sperm motility after freeze-thawing, but DNA damage was neither related to functional membrane integrity nor viable sperm with intact acrosomes. We concluded that DNA fragmentation and loss of motility during cryopreservation of

  12. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity.

    Science.gov (United States)

    Rahiminia, Tahereh; Hosseini, Akram; Anvari, Morteza; Ghasemi-Esmailabad, Saeed; Talebi, Ali Reza

    2017-08-01

    Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial. Normal sample were collected according to WHO strict criteria. Sperm suspensions were mixed 1:1 with 0.5 M sucrose and divided into four equal aliquots for freezing: fresh, nitrogen direct immersion vitrification (Vit), solid surface vitrification (SSV) and in vapour (Vapour). Sperm suspensions were transferred into a 0.25 ml sterile plastic. Then straw was inserted inside the 0.5 ml straw. For thawing, the straws were immersed in a 42 °C water bath. Beside the sperm parameters, we assessed the acrosome reaction by double staining, chromatin integrity by toluidine blue (Tb) and chromomycin A3 (CMA3) and DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) respectively. In progressive motility, the highest rate occurred in Vit (39.9 ± 13.3). Moreover, the lowest rate of immotile sperm was in Vit (32.7 ± 16.3). In normal morphology, the group Vit was similar to the fresh, while SSV and Vapour were significantly different from the fresh. The percentage of acrosome-reacted sperms was more in Vit (81.3 ± 10.2) than the fresh group. TUNEL+ results showed that DNA fragmentation was significantly increased in Vit (p-value = 0.025). While in SSV and Vapour results were comparable to fresh. There was a significant correlation between TUNEL+ and normal morphology, TB, CMA3 and presence of intact acrosome. Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome

  13. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

    Science.gov (United States)

    Ito, Takuji; Bai, Tao; Tanaka, Tetsuji; Yoshida, Kenji; Ueyama, Takashi; Miyajima, Masayasu; Negishi, Takayuki; Kawasaki, Takahiko; Takamatsu, Hyota; Kikutani, Hitoshi; Kumanogoh, Atsushi; Yukawa, Kazunori

    2015-02-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The

  14. Gaseous signalling molecule SO2 via Hippo‑MST pathway to improve myocardial fibrosis of diabetic rats.

    Science.gov (United States)

    Liu, Maojun; Liu, Shengquan; Tan, Wenting; Tang, Fen; Long, Junrong; Li, Zining; Liang, Biao; Chu, Chun; Yang, Jun

    2017-12-01

    Recent studies have indicated the existence of an endogenous sulfur dioxide (SO2)‑generating system in the cardiovascular system. The present study aimed to discuss the function and regulatory mechanism of gaseous signal molecule SO2 in inhibiting apoptosis and endoplasmic reticulum stress (ERS) via the Hippo‑MST signaling pathway to improve myocardial fibrosis of diabetic rats. A total of 40 male Sprague‑Dawley rats were randomly divided into four groups (10 rats per group): Normal control group (control group), diabetic rats group [streptozotocin (STZ) group], SO2 intervention group (STZ+SO2 group) and diabetes mellitus rats treated with L‑Aspartic acid β‑hydroxamate (HDX) group (HDX group). Diabetic rats models were established by intra‑peritoneal injection of STZ (40 mg/kg) Following model establishment, intra‑peritoneal injection of Na2SO3/NaHSO3 solution (0.54 mmol/kg) was administered in the STZ+SO2 group, and HDX solution (25 mg/kg/week) was administered in the HDX group. A total of 4 weeks later, echocardiography was performed to evaluate rats' cardiac function; Masson staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and transmission electron microscopy examinations were performed to observe myocardial morphological changes. ELISA was employed to determine the SO2 content. Western blot analysis was performed to detect the expression of proteins associated with apoptosis, ERS and the Hippo‑MST signalling pathway. Compared with the control group, the STZ group and HDX group had a disordered arrangement of myocardial cells with apparent myocardial fibrosis, and echocardiography indicated that the cardiac function was lowered, there was an obvious increase of apoptosis in myocardial tissue, the expression levels of apoptosis‑associated protein B‑cell lymphoma associated protein X, caspase‑3 and caspase‑9 were upregulated, and Bcl‑2 expression was downregulated. The expression of ERS and Hippo

  15. Glycogen synthase kinase-3: a key kinase in retinal neuron apoptosis in early diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    Li Zhaohui; Ma Ling; Chen Xiaodong; Li Yonghao; Li Shiyi; Zhang Jinglin; Lu Lin

    2014-01-01

    Background Diabetes-related pathogenic factors can cause retinal ganglion cell (RGC) apoptosis,but the specific mechanism is not very clear.The aim of this study is to investigate the correlation between glycogen synthase kinase-3 (GSK-3) activation and retinal neuron apoptosis.Methods In an in vitro experiment,the number of apoptotic RGC-5 cells differentiated by staurosporine was evaluated via flow cytometry and nuclei staining using Hoechst 33258.GSK-3 phosphorylation and caspase-3 activation in RGC-5 cells after serum deprivation were determined using Western blotting.Mitochondrial membrane potential was detected using the dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetrethyl benzimidalyl carbocyanine iodide,and reactive oxygen species (ROS) levels were measured with dihydroethidium.In an in vivo experiment,the number of apoptotic retinal neurons was evaluated via terminal transferase dUTP nick-end labeling (TUNEL),and GSK-3 phosphorylation was determined using Western blotting,in the retinal nerve epithelial tissue of rats in which diabetes was induced by intravenous tail-vein injection of streptozotocin for 4 weeks.Results The levels of phosphorylated Ser21/9 in GSK-3α/β and p-T308/S473-AKT were lower and the cleaved caspase-3 levels were higher in the serum-deprived model (P <0.05).Lithium chloride treatment was associated with a slower rate of apoptosis,increased mitochondrial membrane potential,and decreased ROS levels in differentiated RGC-5 cells (P <0.05).The level of blood glucose and the number of TUNEL-positive cells in the whole-mounted retinas were higher (P <0.01),and the levels of phosphorylated Ser21/9 in GSK-3α/β and body weight were lower (P <0.05).However,the thickness of the retinal nerve epithelial layer was not significantly less in diabetic rats compared with control group.Lithium chloride intravitreal injection increased the levels of phosphorylated Ser21/9 in GSK-3α/β and decreased TUNEL-positive cells in the whole-mounted retinas

  16. The effectiveness of 125I seed interstitial brachytherapy for transplantation tumor of human pancreatic carcinoma in nude mice: an experiment in vivo

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    Song Qi; Liu Yu; Wang Zhongmin; Huang Wei; Lu Jian; Chen Kemin

    2010-01-01

    Objective: To discuss the effectiveness and therapeutic mechanism of 125 I interstitial brachytherapy for transplantation tumor of human pancreatic carcinoma in nude mice. Methods: The human pancreatic cell line Sw1990 was subcutaneously injected into the right lower limb partially dorsal area next to the groin of the immunodeficient BABL /c nude mice. The tumor was removed and cut into small pieces after it was formed,then the tumor pieces were inoculated in nude mice. The tumor developed to 8-10 mm in size after six weeks. A total of 16 nude mice with the suitable tumor size were used in this study. The 16 experimental mice were randomly and equally divided into two groups. The mice in study group (n = 8) were implanted with 125 I seeds, while the mice in control group (n = 8) were implanted with ghost seeds. After the implantation both the long and short diameter of the tumors as well as the mouse body weight were measured every 4 days. The tumor weight was measured when the mouse was sacrificed. The paraffin-embedded samples were sent for histopathological examination. Apoptotic cells were checked with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Expression of proliferating cell nuclear antigen (PCNA) was detected with immuno-histochemical staining. Results: The tumor grew slowly in the study group, but rapidly in the control group. The tumor weight in the study group and the control group was (2.68 ± 0.70)g and (4.68 ± 1.45)g, respectively, the difference between two groups was statistically significant (P = 0.021). The tumor inhibition rate was about 42.66%. No significant difference in body weight of nude mice existed between two groups both before and after the treatment (P > 0.05). Marked tumor necrosis was seen in study group, but no obvious, or only a little, tumor necrosis could be observed in the control group. The apoptotic index checked with the TUENL method in the study group and control group was (23.2 ± 1.9)% and

  17. In vitro investigation of the effect of matrix molecules on the behavior of colon cancer cells under the effect of geldanamycin derivative.

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    Vural, Kamil; Kosova, Funda; Kurt, Feyzan Özdal; Tuğlu, İbrahim

    2017-10-01

    The chaperone-binding drug, 17-allylamino-17-demethoxygeldanamycin, has recently come into clinical use. It is a derivative of geldanamycin, an ansamycin benzoquinone antibiotic with anti-carcinogenic effect. Understanding the effect of this drug on the cancer cells and their niche is important for treatment. We applied 17-allylamino-17-demethoxygeldanamycin to colon cancer cell line (Colo 205) on matrix molecules to investigate the relationship of apoptosis with terminal deoxynucleotidyl transferase dUTP nick end labeling immunocytochemistry and related gene expression. We used laminin and collagen I for matrix molecules and vascular endothelial growth factor for angiogenic structure. We also examined apoptosis-related signaling pathway including mitochondrial proteins, cytochrome c, Bcl-2, caspase-9, Apaf-1 expression using real-time polymerase chain reaction. There was clear effect of 17-allylamino-17-demethoxygeldanamycin that killed more cells on tissue culture plastic compared to matrix molecules. The IC 50 value was 0.58 µg/mL for tissue culture plastic compared with 0.64 µg/mL for laminin and 0.75 µg/mL for collagen I. The analyses showed that more cells on matrix molecules underwent apoptosis compared to that on tissue culture plastic. Apoptosis-related gene expression was similar in which Bcl-2 expression decreased and proapoptotic gene expression of the cells on matrix molecules increased compared to that on tissue culture plastic. However, the application of 17-allylamino-17-demethoxygeldanamycin was more effective for the cells on collagen I compared to the cells on laminin. There was also a decrease in angiogenesis as shown by the vascular endothelial growth factor staining. This was more pronounced by coating of the tissue culture plastic with matrix molecules. Our results supported the anti-cancer effect of 17-allylamino-17-demethoxygeldanamycin, and this effect depended on matrix molecules. This effect occurs through apoptosis, and related

  18. Double suicide genes selectively kill human umbilical vein endothelial cells

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    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  19. Clusterin Reduces Cold Ischemia-Reperfusion Injury in Heart Transplantation Through Regulation of NF-kB Signaling and Bax/Bcl-xL Expression

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    Guodong Liu

    2018-02-01

    Full Text Available Background/Aims: Ischemia-reperfusion (I/R injury is an unavoidable event occurring during heart transplantation and is a key factor in graft failure and the long-term survival rate of recipients. Therefore, there is an urgent need for the development of new therapies to prevent I/R injury. Clusterin is a hetero-dimeric glycoprotein with an antiapoptotic function. In this study, we investigated whether clusterin was cardioprotective in heart transplantation against I/R injury using an in vivo rat model and an in vitro cell culture system, and examined the underlying mechanisms of I/R injury. Methods: Heart grafts from wild-type C57BL/6 mice were preserved in UW solution (control or UW solution containing recombinant human apolipoprotein-J (hr clusterin for 24 h. The preserved hearts were implanted into recipient mice of the same strain as the donors for 72 h, and the heart grafts were then taken for histopathological and gene expression analyses. An in vitro ischemia reperfusion model using H9C2 cells or H9C2/clusterin cDNA cells was constructed. The expression of clusterin, p65, Bax, Bcl-xL, IL-1β, and TNF-α protein and mRNA in heart tissue and H9C2 cells was detected by western blot, reverse transcription-polymerase chain reaction (RT-PCR, and quantitative RT-PCR assays; IL-1β and TNF-α protein was detected by enzyme-linked immunosorbent assays; NF-kB activity was detected by an electrophoretic mobility shift assay; cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometric analyses. Results: Cold I/R caused severe morphologic myocardial injury to heart grafts from wild-type C57BL/6 mice, whereas grafts from hr clusterin preservation showed less damage, as demonstrated by decreased cell apoptosis/death, decreased neutrophil infiltration, and the preservation of the normal structure of the heart. Clusterin reduced the expression of p65, pre-inflammatory IL-1β, and TNF-α, and

  20. Apoptosis is increased and cell proliferation is decreased in out-of-phase endometria from infertile and recurrent abortion patients

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    Irigoyen Marcela

    2010-10-01

    Full Text Available Abstract Background Various endometrial abnormalities have been associated with luteal phase deficiency: a significant dyssynchrony in the maturation of the glandular epithelium and the stroma and a prevalence of out-of-phase endometrial biopsy specimens. Out-of phase endometrium is a controversial disorder related to failed implantation, infertility and early pregnancy loss. Given that the regulation of the apoptotic process in endometrium of luteal phase deficiency is still unknown, the aim of this study was to evaluate cell proliferation, apoptosis and the levels of the main effector caspase, caspase-3 in the luteal in-phase and out-of-phase endometrium. Methods Thirty-seven endometrial samples from sterile or recurrent abortion patients were included in this study: 21 in-phase samples (controls and 16 samples with out-of-phase endometrium. Biopsy specimens of eutopic endometrium were obtained from all subjects during days 21-25 of the menstrual cycle. The endometrium with endometrial maturity of cycle day 25 or less at the time of menstruation was considered out-of phase. Endometrial tissues were fixed in 10% buffered formaldehyde. For apoptosis quantification, sections were processed for in situ immunohistochemical localization of nuclei exhibiting DNA fragmentation, by the terminal deoxynucleotidyl transferase (TdT-mediated dUTP digoxygenin nick-end labeling (TUNEL technique. Expressions of Proliferating Cell Nuclear Antigen (PCNA as a marker of cell proliferation, and of cleaved caspase-3 as a marker of apoptosis, were assessed by immunohistochemistry in the luteal in-phase and out-of-phase endometrium from infertile and recurrent abortion patients. Results Luteal out-of-phase endometrium had increased apoptosis levels compared to in-phase endometrium (p Conclusions this study represents the first report describing variations at the cell proliferation and cell death levels in the out-of-phase endometrium in comparison with in

  1. Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic

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    An S

    2013-02-01

    Full Text Available Songhie An,* Kihoon Nam,* Sunghyun Choi, Cheng Z Bai, Yan Lee, Jong-Sang ParkDepartment of Chemistry, Seoul National University, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model.Methods and results: The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4. Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of >40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay, 4´,6-Diamidino-2-phenylindole (DAPI TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining.Conclusion: The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier's high transfection efficiency (35%–40% in glioma cells and the selective apoptosis-inducing activity of

  2. Effect of superoxide anion scavenger on rat hearts with chronic intermittent hypoxia.

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    Pai, Peiying; Lai, Ching Jung; Lin, Ching-Yuang; Liou, Yi-Fan; Huang, Chih-Yang; Lee, Shin-Da

    2016-04-15

    Only very limited information regarding the protective effects of the superoxide anion scavenger on chronic intermittent hypoxia-induced cardiac apoptosis is available. The purpose of this study is to evaluate the effects of the superoxide anion scavenger on cardiac apoptotic and prosurvival pathways in rats with sleep apnea. Forty-two Sprague-Dawley rats were divided into three groups, rats with normoxic exposure (Control, 21% O2, 1 mo), rats with chronic intermittent hypoxia exposure (Hypoxia, 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo), and rats with pretreatment of the superoxide anion scavenger and chronic intermittent hypoxia exposure (Hypoxia-O2 (-)-Scavenger, MnTMPyP pentachloride, 1 mg/kg ip per day; 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo) at 5-6 mo of age. After 1 mo, the protein levels and apoptotic cells of excised hearts from three groups were measured by Western blotting and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay. The superoxide anion scavenger decreased hypoxia-induced myocardial architecture abnormalities, left ventricular hypertrophy, and TUNEL-positive apoptosis. The superoxide anion scavenger decreased hypoxia-induced Fas ligand, Fas death receptors, Fas-associated death domain (FADD), activated caspase-8, and activated caspase-3 (Fas-dependent apoptotic pathway) as well as Bad, activated caspase-9 and activated caspase-3 (mitochondria-dependent apoptotic pathway), endonuclease G (EndoG), apoptosis-inducing factor (AIF), and TUNEL-positive apoptosis. The superoxide anion scavenger increased IGF-1, IGF-1R, p-PI3k, p-Akt, p-Bad, Bcl-2, and Bcl-xL (survival pathway). Our findings imply that the superoxide anion scavenger might prevent cardiac Fas-mediated and mitochondrial-mediated apoptosis and enhance the IGF-1-related survival pathway in chronic intermittent hypoxia. The superoxide anion scavenger may prevent chronic sleep apnea-enhanced cardiac apoptotic pathways and enhances

  3. Adiponectin protects rat myocardium against chronic intermittent hypoxia-induced injury via inhibition of endoplasmic reticulum stress.

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    Wenxiao Ding

    Full Text Available Obstructive sleep apnea syndrome (OSAS is associated with many cardiovascular disorders such as heart failure, hypertension, atherosclerosis, and arrhythmia and so on. Of the many associated factors, chronic intermittent hypoxia (CIH in particular is the primary player in OSAS. To assess the effects of CIH on cardiac function secondary to OSAS, we established a model to study the effects of CIH on Wistar rats. Specifically, we examined the possible underlying cellular mechanisms of hypoxic tissue damage and the possible protective role of adiponectin against hypoxic insults. In the first treatment group, rats were exposed to CIH conditions (nadir O2, 5-6% for 8 hours/day, for 5 weeks. Subsequent CIH-induced cardiac dysfunction was measured by echocardiograph. Compared with the normal control (NC group, rats in the CIH-exposed group experienced elevated levels of left ventricular end-systolic dimension and left ventricular end-systolic volume and depressed levels of left ventricular ejection fraction and left ventricular fractional shortening (p<0.05. However, when adiponectin (Ad was added in CIH + Ad group, we saw a rescue in the elevations of the aforementioned left ventricular function (p<0.05. To assess critical cardiac injury, we detected myocardial apoptosis by Terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling (TUNEL analysis. It was showed that the apoptosis percentage in CIH group (2.948% was significantly higher than that in NC group (0.4167% and CIH + Ad group (1.219% (p<0.05. Protein expressions of cleaved caspase-3, cleaved caspase-9, and cleaved-caspase-12 validated our TUNEL results (p<0.05. Mechanistically, our results demonstrated that the proteins expressed with endoplasmic reticulum stress and the expression of reactive oxygen species (ROS were significantly elevated under CIH conditions, whereas Ad supplementation partially decreased them. Overall, our results suggested that Ad augmentation could improv