Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line.

PubMed

Shirata, Noriko; Ikeda, Motoko; Kobayashi, Michihiro

2010-03-15

We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuDeltaep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuDeltaep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV. Copyright 2009 Elsevier Inc. All rights reserved.

Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection

PubMed Central

Shang, Shaobin; Siddiqui, Sarah; Bian, Yao; Zhao, Jie; Wang, Chyung-Ru

2016-01-01

MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules. PMID:27272249

Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.

PubMed

Müller, Marcel A; Raj, V Stalin; Muth, Doreen; Meyer, Benjamin; Kallies, Stephan; Smits, Saskia L; Wollny, Robert; Bestebroer, Theo M; Specht, Sabine; Suliman, Tasnim; Zimmermann, Katrin; Binger, Tabea; Eckerle, Isabella; Tschapka, Marco; Zaki, Ali M; Osterhaus, Albert D M E; Fouchier, Ron A M; Haagmans, Bart L; Drosten, Christian

2012-12-11

A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is highly critical, because the SARS-CoV receptor influenced pathology, and its localization in the deep respiratory tract is thought to have restricted the transmissibility of SARS. Our data show that hCoV-EMC does not need the SARS-CoV receptor to infect human cells. Moreover, the virus is capable of infecting human, pig, and bat cells. This is remarkable, as human CoVs normally cannot replicate in bat cells as a consequence of host adaptation. Our results implicate that the new virus might use a receptor that is conserved between bats, pigs and humans suggesting a low barrier against cross-host transmission.

Tamarix gallica phenolics protect IEC-6 cells against H2O2 induced stress by restricting oxidative injuries and MAPKs signaling pathways.

PubMed

Bettaib, Jamila; Talarmin, Hélène; Droguet, Mickaël; Magné, Christian; Boulaaba, Mondher; Giroux-Metges, Marie-Agnès; Ksouri, Riadh

2017-05-01

Polyphenolic compounds gained interest in the pharmaceutical research area due to their beneficial properties. Herein, antioxidant and cytoprotective capacities of T. gallica extract on H 2 O 2 -challenged rat small intestine epithelial cells were investigated. To set stress conditions, IEC-6 cultures were challenged with numerous H 2 O 2 doses and durations. Then, 40μM H 2 O 2 during 4h were selected to assess the cytoprotective effect of different T. gallica extract concentrations. Oxidative parameters, measured through CAT and SOD activities as well as MDA quantification were assessed. In addition, the expression of possibly involved MAPKs was also valued. Main results reported that T. gallica was rich in polyphenols and exhibited an important antioxidant activity (DPPH Assay, IC 50 =6μgmL -1 ; ABTS + test, IC 50 =50μgmL -1 ; Fe-reducing power, EC 50 =100μgmL -1 ). The exposure of IEC-6 cultures to 40μM H 2 O 2 during 4h caused oxidative stress manifested by (i) over 70% cell mortality, (ii) over-activity of CAT (246%), (iii) excess in MDA content (18.4nmolmg -1 ) and (iiii) a trigger of JNK phosphorylation. Pretreatment with T. gallica extract, especially when used at 0.25μgmL -1 , restored cell viability to 122%, and normal cell morphology in H 2 O 2 -chalenged cells. In addition, this extract normalized CAT activity and MDA content (100% and 14.7nmolmg -1 , respectively) to their basal levels as compared to control cells. Furthermore, stopping cell death seems to be due to dephosphorylated JNK MAPK exerted by T. gallica bioactive compounds. In all, T. gallica components provided a cross-talk between regulatory pathways leading to an efficient cytoprotection against harmful oxidative stimulus. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Effect of restriction vegan diet's on muscle mass, oxidative status, and myocytes differentiation: A pilot study.

PubMed

Vanacore, Daniela; Messina, Giovanni; Lama, Stefania; Bitti, Giuseppe; Ambrosio, Pasqualina; Tenore, Giancarlo; Messina, Antonietta; Monda, Vincenzo; Zappavigna, Silvia; Boccellino, Mariarosaria; Novellino, Ettore; Monda, Marcellino; Stiuso, Paola

2018-01-10

This study was conceived to evaluate the effects of three different diets on body composition, metabolic parameters, and serum oxidative status. We enrolled three groups of healthy men (omnivores, vegetarians, and vegans) with similar age, weight and BMI, and we observed a significant decrease in muscle mass index and lean body mass in vegan compared to vegetarian and omnivore groups, and higher serum homocysteine levels in vegetarians and vegans compared to omnivores. We studied whether serum from omnivore, vegetarian, and vegan subjects affected oxidative stress, growth and differentiation of both cardiomyoblast cell line H9c2 and H-H9c2 (H9c2 treated with H 2 O 2 to induce oxidative damage). We demonstrated that vegan sera treatment of both H9c2 and H-H9c2 cells induced an increase of TBARS values and cell death and a decrease of free NO 2- compared to vegetarian and omnivorous sera. Afterwards, we investigated the protective effects of vegan, vegetarian, and omnivore sera on the morphological changes induced by H 2 O 2 in H9c2 cell line. We showed that the omnivorous sera had major antioxidant and differentiation properties compared to vegetarian and vegan sera. Finally, we evaluated the influence of the three different groups of sera on MAPKs pathway and our data suggested that ERK expression increased in H-H9c2 cells treated with vegetarian and vegan sera and could promote cell death. The results obtained in this study demonstrated that restrictive vegan diet could not prevent the onset of metabolic and cardiovascular diseases nor protect by oxidative damage. © 2018 Wiley Periodicals, Inc.

Inhibition of Avian Influenza A Virus Replication in Human Cells by Host Restriction Factor TUFM Is Correlated with Autophagy.

PubMed

Kuo, Shu-Ming; Chen, Chi-Jene; Chang, Shih-Cheng; Liu, Tzu-Jou; Chen, Yi-Hsiang; Huang, Sheng-Yu; Shih, Shin-Ru

2017-06-13

Avian influenza A viruses generally do not replicate efficiently in human cells, but substitution of glutamic acid (Glu, E) for lysine (Lys, K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses, host restriction factors associated with PB2 627 E have yet to be fully investigated. We conducted immunoprecipitation, followed by differential proteomic analysis, to identify proteins associating with PB2 627 K (human signature) and PB2 627 E (avian signature) of influenza A/WSN/1933(H1N1) virus, and the results indicated that Tu elongation factor, mitochondrial (TUFM), had a higher binding affinity for PB2 627 E than PB2 627 K in transfected human cells. Stronger binding of TUFM to avian-signature PB2 590 G/ 591 Q and PB2 627 E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2 627 E demonstrated increased replication in TUFM-deficient cells, but viral replication decreased in cells overexpressing TUFM. Interestingly, the presence of TUFM specifically inhibited the replication of PB2 627 E viruses, but not PB2 627 K viruses. In addition, enhanced levels of interaction between TUFM and PB2 627 E were noted in the mitochondrial fraction of infected cells. Furthermore, TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2 627 E virus; however, autophagy remained consistent in PB2 627 K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy. IMPORTANCE An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both critical to the prevention and control of emerging viruses that cross species barriers to target new hosts. Using a proteomic approach, we revealed a novel role for TUFM as a host restriction factor that exerts an inhibitory effect on avian-signature PB2 627 E influenza virus propagation in human cells. We further found that increased TUFM-dependent autophagy correlates with the inhibitory effect on avian-signature influenza virus replication and may serve as a key intrinsic mechanism to restrict avian influenza virus infection in humans. These findings provide new insight regarding the TUFM mitochondrial protein and may have important implications for the development of novel antiviral strategies. Copyright © 2017 Kuo et al.

The genetic origin of minor histocompatibility antigens.

PubMed

Roopenian, D C; Christianson, G J; Davis, A P; Zuberi, A R; Mobraaten, L E

1993-01-01

The purpose of this study was to elucidate the genetic origin of minor histocompatibility (H) antigens. Toward this end common inbred mouse strains, distinct subspecies, and species of the subgenus Mus were examined for expression of various minor H antigens. These antigens were encoded by the classical minor H loci H-3 and H-4 or by newly identified minor H antigens detected as a consequence of mutation. Both minor H antigens that stimulate MHC class I-restricted cytotoxic T cells (Tc) and antigens that stimulate MHC class II-restricted helper T cells (Th) were monitored. The results suggested that strains of distinct ancestry commonly express identical or cross-reactive antigens. Moreover, a correlation between the lack of expression of minor H antigens and ancestral heritage was observed. To address whether the antigens found on unrelated strains were allelic with the sensitizing minor H antigens or a consequence of antigen cross-reactivity, classical genetic segregation analysis was carried out. Even in distinct subspecies and species, the minor H antigens always mapped to the site of the appropriate minor H locus. Together the results suggest: 1) minor H antigen sequences are evolutionarily stable in that their pace of antigenic change is slow enough to predate subspeciation and speciation; 2) the minor H antigens originated in the inbred strains as a consequence of a rare polymorphism or loss mutation carried in a founder mouse stock that caused the mouse to perceive the wild-type protein as foreign; 3) there is a remarkable lack of antigenic cross-reactivity between the defined minor H antigens and other gene products.

Absence of Nrf2 or Its Selective Overexpression in Neurons and Muscle Does Not Affect Survival in ALS-Linked Mutant hSOD1 Mouse Models

PubMed Central

Vargas, Marcelo R.; Burton, Neal C.; Gan, Li; Johnson, Delinda A.; Schäfer, Matthias; Werner, Sabine; Johnson, Jeffrey A.

2013-01-01

The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1G93A mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS. PMID:23418589

Absence of Nrf2 or its selective overexpression in neurons and muscle does not affect survival in ALS-linked mutant hSOD1 mouse models.

PubMed

Vargas, Marcelo R; Burton, Neal C; Kutzke, Jennifer; Gan, Li; Johnson, Delinda A; Schäfer, Matthias; Werner, Sabine; Johnson, Jeffrey A

2013-01-01

The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1(G93A) mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.

Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing

PubMed Central

1983-01-01

We examined the ability of a set of cloned chicken ovalbumin (cOVA)- specific, Id-restricted, T cell hybridomas to produce interleukin-2 in response to cOVA presented by the Ia+ B cell lymphoma line, A20-2J. Although viable A20-2J cells presented native, denatured, and fragmented cOVA more or less equally well, A20-2J cells that were glutaraldehyde-fixed could present only enzymatically or chemically fragmented cOVA. These results suggest that antigen fragmentation may be both necessary and sufficient to define accessory cell processing of soluble antigens so that they may be recognized in association with I- region molecules by T cells. PMID:6193218

A Quantitative Increase in Regulatory T Cells Controls Development of Vitiligo

PubMed Central

Chatterjee, Shilpak; Eby, Jonathan; Al-Khami, Amir A.; Soloshchenko, Myroslawa; Kang, Hee-Kap; Kaur, Navtej; Naga, Osama; Murali, Anuradha; Nishimura, Michael I.; Le Poole, I. Caroline; Mehrotra, Shikhar

2014-01-01

T cell cytolytic activity targeting epidermal melanocyte is shown to cause progressive depigmentation and autoimmune vitiligo. Using the recently developed transgenic mice h3TA2 that carry T cell with a HLA-A2 restricted human tyrosinase reactive TCR and develop spontaneous vitiligo from an early age, we addressed the mechanism regulating autoimmune vitiligo. Depigmentation was significantly impaired only in IFN-γ knockout h3TA2 mice but not in TNF-α or perforin knockout h3TA2 mouse strains, confirming a central role for IFN-γ in vitiligo development. Additionally, the regulatory T cells (Treg) were relatively abundant in h3TA2-IFN-γ−/− mice, and depletion of Treg employing anti-CD25 antibody fully restored the depigmentation phenotype in h3TA2-IFN-γ−/− mice mediated in part through upregulation of pro-inflammatory cytokines as IL-17and IL-22. Further therapeutic potential of Treg abundance in preventing progressive depigmentation was evaluated by adoptively transferring purified Treg or using rapamycin. Both adoptive transfer of Treg and rapamycin induced lasting remission of vitiligo in mice treated at the onset of disease, or in mice with established disease. This leads us to conclude that reduced regulatory responses are pivotal to the development of vitiligo in disease-prone mice, and that a quantitative increase in the Treg population may be therapeutic for vitiligo patients with active disease. PMID:24366614

DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells

PubMed Central

Laos, Maarja; Anttonen, Tommi; Kirjavainen, Anna; Hällström, Taija af; Laiho, Marikki; Pirvola, Ulla

2014-01-01

Supporting cells (SCs) of the cochlear (auditory) and vestibular (balance) organs hold promise as a platform for therapeutic regeneration of the sensory hair cells. Prior data have shown proliferative restrictions of adult SCs forced to re-enter the cell cycle. By comparing juvenile and adult SCs in explant cultures, we have here studied how proliferative restrictions are linked with DNA damage signaling. Cyclin D1 overexpression, used to stimulate cell cycle re-entry, triggered higher proliferative activity of juvenile SCs. Phosphorylated form of histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) were induced in a foci-like pattern in SCs of both ages as an indication of DNA double-strand break formation and activated DNA damage response. Compared to juvenile SCs, γH2AX and the repair protein Rad51 were resolved with slower kinetics in adult SCs, accompanied by increased apoptosis. Consistent with the in vitro data, in a Rb mutant mouse model in vivo, cell cycle re-entry of SCs was associated with γH2AX foci induction. In contrast to cell cycle reactivation, pharmacological stimulation of SC-to-hair-cell transdifferentiation in vitro did not trigger γH2AX. Thus, DNA damage and its prolonged resolution are critical barriers in the efforts to stimulate proliferation of the adult inner ear SCs. PMID:25063730

Arenaviruses. Genes, proteins, and expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oldstone, M.B.A.

1987-01-01

This book provides a discussion of current knowledge on Arenaviruses. These viruses are the cause of major health problems, such as Lassa fever and Junin virus disease, and have been the Rosetta stone on which many of the major concepts in viral pathogenesis and immunobiology have been built. For example, study of lymphocytic choriomeningitis naturally and experimentally induced infection in the normal mouse host presented the scientific community with the first and definitive work on the following topics: virus induced immune response disease, immunologic tolerance, virus induced immune complex disease, presence and generation of cytotoxic T cells in vitro andmore » in vivo, H-2 restriction and dual recognition phenomena, and viral disease induced by altering physiologic or differential functions of a cell without causing alterations of house keeping or vital functions, i.e. pathology in the absence of cell or tissue lysis.« less

H3S10ph broadly marks early-replicating domains in interphase ESCs and shows reciprocal antagonism with H3K9me2.

PubMed

Chen, Carol C L; Goyal, Preeti; Karimi, Mohammad M; Abildgaard, Marie H; Kimura, Hiroshi; Lorincz, Matthew C

2018-01-01

Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants . Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp ) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1 -/- and Ehmt2 -/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells. © 2018 Chen et al.; Published by Cold Spring Harbor Laboratory Press.

Neoplastic cell transformation by high-LET radiation - Molecular mechanisms

NASA Technical Reports Server (NTRS)

Yang, Tracy Chui-Hsu; Craise, Laurie M.; Tobias, Cornelius A.; Mei, Man-Tong

1989-01-01

Quantitative data were collected on dose-response curves of cultured mouse-embryo cells (C3H10T1/2) irradiated with heavy ions of various charges and energies. Results suggests that two breaks formed on DNA within 80 A may cause cell transformation and that two DNA breaks formed within 20 A may be lethal. From results of experiments with restriction enzymes which produce DNA damages at specific sites, it was found that DNA double strand breaks are important primary lesions for radiogenic cell transformation and that blunt-ended double-strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship for high-LET radiation is similar to that for HGPRT locus mutation, chromosomal deletion, and cell transformation, indicating that common lesions may be involved in these radiation effects.

The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication

PubMed Central

Aparicio, Tomás; Guillou, Emmanuelle; Coloma, Javier; Montoya, Guillermo; Méndez, Juan

2009-01-01

The GINS complex, originally discovered in Saccharomyces cerevisiae and Xenopus laevis, binds to DNA replication origins shortly before the onset of S phase and travels with the replication forks after initiation. In this study we present a detailed characterization of the human GINS (hGINS) homolog. Using new antibodies that allow the detection of endogenous hGINS in cells and tissues, we have examined its expression, abundance, subcellular localization and association with other DNA replication proteins. Expression of hGINS is restricted to actively proliferating cells. During the S phase, hGINS becomes part of a Cdc45–MCM–GINS (CMG) complex that is assembled on chromatin. Down-regulation of hGINS destabilizes CMG, causes a G1–S arrest and slows down ongoing DNA replication, effectively blocking cell proliferation. Our data support the notion that hGINS is an essential component of the human replisome. PMID:19223333

Ara h 2 peptides containing dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic.

PubMed

Prickett, Sara R; Voskamp, Astrid L; Dacumos-Hill, April; Symons, Karen; Rolland, Jennifer M; O'Hehir, Robyn E

2011-03-01

Peanut allergy is a life-threatening condition; there is currently no cure. Although whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy. This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4(+) T cells but do not bind IgE as candidates for safe peanut-specific immunotherapy. Multiple CD4(+) T-cell lines specific for the major peanut allergen Ara h 2 were generated from PBMCs of 16 HLA-diverse subjects with peanut allergy by using 5,6-carboxyfluorescein diacetate succinimidylester-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA restriction of core epitope recognition was investigated by using anti-HLA blocking antibodies and HLA genotyping. Serum-IgE peptide-binding was assessed by dot-blot. Five dominant CD4(+) T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, HLA-DP, and HLA-DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants containing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE, and could directly target T(H)2-type T cells in peripheral blood of subjects with allergy. Short CD4(+) T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell-targeted peanut-specific immunotherapy for an HLA-diverse population. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Loss of retrovirus production in JB/RH melanoma cells transfected with H-2Kb and TAP-1 genes.

PubMed

Li, M; Xu, F; Muller, J; Huang, X; Hearing, V J; Gorelik, E

1999-01-20

JB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kb molecules due to down-regulation of the H-2Kb and TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kb and cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kb molecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviral PstI restriction site and the loss of a HindIII restriction site in the pol region. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA. Copyright 1999 Academic Press.

5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin

2014-08-08

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562more » cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.« less

Cidofovir is active against human papillomavirus positive and negative head and neck and cervical tumor cells by causing DNA damage as one of its working mechanisms.

PubMed

Mertens, Barbara; Nogueira, Tatiane; Stranska, Ruzena; Naesens, Lieve; Andrei, Graciela; Snoeck, Robert

2016-07-26

Human papillomavirus (HPV) causes cervical cancer and a large fraction of head and neck squamous cell carcinomas (HNSCC). Cidofovir (CDV) proved efficacious in the treatment of several HPV-induced benign and malignant hyper proliferations. To provide a better insight into how CDV selectively eradicates transformed cells, HPV+ and HPV- cervical carcinoma and HNSCC cell lines were compared to normal cells for antiproliferative effects, CDV metabolism, drug incorporation into cellular DNA, and DNA damage. Incorporation of CDV into cellular DNA was higher in tumor cells than in normal cells and correlated with CDV antiproliferative effects, which were independent of HPV status. Increase in phospho-ATM levels was detected following CDV exposure and higher levels of γ-H2AX (a quantitative marker of double-strand breaks) were measured in tumor cells compared to normal cells. A correlation between DNA damage and CDV incorporation into DNA was found but not between DNA damage and CDV antiproliferative effects. These data indicate that CDV antiproliferative effects result from incorporation of the drug into DNA causing DNA damage. However, the anti-tumor effects of CDV cannot be exclusively ascribed to DNA damage. Furthermore, CDV can be considered a promising broad spectrum anti-cancer agent, not restricted to HPV+ lesions.

Endogenous APOBEC3B restricts LINE-1 retrotransposition in transformed cells and human embryonic stem cells.

PubMed

Wissing, Silke; Montano, Mauricio; Garcia-Perez, Jose Luis; Moran, John V; Greene, Warner C

2011-10-21

Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ∼2-3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells.

Ethanol-induced impairment of polyamine homeostasis – A potential cause of neural tube defect and intrauterine growth restriction in fetal alcohol syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haghighi Poodeh, Saeid, E-mail: saeid.haghighi@oulu.fi; Medical Research Center, Oulu University Hospital, Oulu; Alhonen, Leena

Highlights: • Polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. • Alcohol administration perturbs polyamine levels in the tissues with various patterns. • Total absence of polyamines in the embryo head at 9.5 dpc is critical for development. • The deficiency is associated with reduction in endothelial cell sprouting in the head. • Retarded migration of neural crest cells may cause development of neural tube defect. - Abstract: Introduction: Polyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism andmore » uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting. Methods: CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals 8.75 days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5 dpc and polyamine concentrations were determined. Results: No measurable quantity of polyamines could be detected in the embryo head at 9.5 dpc, 12 h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction. Discussion: Our results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5 dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.« less

Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

PubMed Central

Ikizler, Mine R.; Kawaoka, Yoshihiro; Rudenko, Larisa G.; Treanor, John J.; Subbarao, Kanta; Wright, Peter F.

2012-01-01

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates. PMID:22915797

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses

PubMed Central

Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A.; Maines, Taronna R.

2016-01-01

ABSTRACT A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type. PMID:27707929

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

PubMed

Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

2016-12-15

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Differential impact of science policy on subfields of human embryonic stem cell research.

PubMed

Moon, Seongwuk; Cho, Seong Beom

2014-01-01

In this research, we examine how restrictive policy influenced performance in human embryonic stem cell research (hESC) between 1998 and 2008. In previous research, researchers argued whether restrictive policy decreased the performance of stem cell research in some nations, especially in the US. Here, we hypothesize that this policy influenced specific subfields of the hESC research. To investigate the selective policy effects, we categorize hESC research publications into three subfields-derivation, differentiation, and medical application research. Our analysis shows that restrictive policy had different effects on different subfields. In general, the US outperformed in overall hESC research throughout these periods. In the derivation of hESC, however, the US almost lost its competence under restrictive policy. Interestingly, the US scientific community showed prominent resilience in hESC research through international collaboration. We concluded that the US resilience and performance stemmed from the wide breadth of research portfolio of US scientists across the hESC subfields, combined with their strategic efforts to collaborate internationally on derivation research.

Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

PubMed Central

Schüller, Ulrich; Heine, Vivi M.; Mao, Junhao; Kho, Alvin T.; Dillon, Allison K.; Han, Young-Goo; Huillard, Emmanuelle; Sun, Tao; Ligon, Azra H.; Qian, Ying; Ma, Qiufu; Alvarez-Buylla, Arturo; McMahon, Andrew P.; Rowitch, David H.; Ligon, Keith L.

2008-01-01

Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors. PMID:18691547

IFITM3 Restricts Human Metapneumovirus Infection.

PubMed

McMichael, Temet M; Zhang, Yu; Kenney, Adam D; Zhang, Lizhi; Zani, Ashley; Lu, Mijia; Chemudupati, Mahesh; Li, Jianrong; Yount, Jacob S

2018-06-15

Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein Toll-like receptor 7 (TLR7), and that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells.

Wound-induced Oxidative Responses in Mountain Birch Leaves

PubMed Central

RUUHOLA, TEIJA; YANG, SHIYONG

2006-01-01

• Aims The aim of the study was to examine oxidative responses in subarctic mountain birch, Betula pubescens subsp. czerepanovii, induced by herbivory and manual wounding. • Methods Herbivory-induced changes in polyphenoloxidase, peroxidase and catalase activities in birch leaves were determined. A cytochemical dye, 3,3-diaminobenzidine, was used for the in situ and in vivo detection of H2O2 accumulation as a response to herbivory and wounding. To localize peroxidase activity in leaves, 10 mm H2O2 was applied to the dye reagent. • Key Results Feeding by autumnal moth, Epirrita autumnata, larvae caused an induction in polyphenoloxidase and peroxidase activities within 24 h, and a concomitant decrease in the activity of antioxidative catalases in wounded leaves. Wounding also induced H2O2 accumulation, which may have both direct and indirect defensive properties against herbivores. Wound sites and guard cells showed a high level of peroxidase activity, which may efficiently restrict invasion by micro-organisms. • Conclusion Birch oxidases together with their substrates may form an important front line in defence against herbivores and pathogens. PMID:16254021

G0-G1 Transition and the Restriction Point in Pancreatic β-Cells In Vivo

PubMed Central

Hija, Ayat; Salpeter, Seth; Klochendler, Agnes; Grimsby, Joseph; Brandeis, Michael; Glaser, Benjamin; Dor, Yuval

2014-01-01

Most of our knowledge on cell kinetics stems from in vitro studies of continuously dividing cells. In this study, we determine in vivo cell-cycle parameters of pancreatic β-cells, a largely quiescent population, using drugs that mimic or prevent glucose-induced replication of β-cells in mice. Quiescent β-cells exposed to a mitogenic glucose stimulation require 8 h to enter the G1 phase of the cell cycle, and this time is prolonged in older age. The duration of G1, S, and G2/M is ∼5, 8, and 6 h, respectively. We further provide the first in vivo demonstration of the restriction point at the G0-G1 transition, discovered by Arthur Pardee 40 years ago. The findings may have pharmacodynamic implications in the design of regenerative therapies aimed at increasing β-cell replication and mass in patients with diabetes. PMID:24130333

Vanishing amplitude of backbone dynamics causes a true protein dynamical transition: H2 NMR studies on perdeuterated C-phycocyanin

NASA Astrophysics Data System (ADS)

Kämpf, Kerstin; Kremmling, Beke; Vogel, Michael

2014-03-01

Using a combination of H2 nuclear magnetic resonance (NMR) methods, we study internal rotational dynamics of the perdeuterated protein C-phycocyanin (CPC) in dry and hydrated states over broad temperature and dynamic ranges with high angular resolution. Separating H2 NMR signals from methyl deuterons, we show that basically all backbone deuterons exhibit highly restricted motion occurring on time scales faster than microseconds. The amplitude of this motion increases when a hydration shell exists, while it decreases upon cooling and vanishes near 175 K. We conclude that the vanishing of the highly restricted motion marks a dynamical transition, which is independent of the time window and of a fundamental importance. This conclusion is supported by results from experimental and computational studies of the proteins myoglobin and elastin. In particular, we argue based on findings in molecular dynamics simulations that the behavior of the highly restricted motion of proteins at the dynamical transition resembles that of a characteristic secondary relaxation of liquids at the glass transition, namely the nearly constant loss. Furthermore, H2 NMR studies on perdeuterated CPC reveal that, in addition to highly restricted motion, small fractions of backbone segments exhibit weakly restricted dynamics when temperature and hydration are sufficiently high.

Elicitation of anti-viral cytotoxic T lymphocytes with purified viral and H-2 antigens.

PubMed

Hale, A H; Ruebush, M J; Harris, D T

1980-07-01

The minimal molecular requirements for elicitation of secondary anti-Sendai virus CTL were investigated. The hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus and the H-2Kk glycoprotein of YAC tumor cells were purified and incorporated into phospholipid vesicles. These unilamellar liposomes were then tested for the ability to elicit H-2 restricted secondary anti-Sendai virus CTL. The results indicate that these well-defined vesicles were capable of eliciting secondary anti-Sendai virus CTL which lysed only target cells possessing the H-2Kk haplotype and modified with inactivated Sendai virus.

Endogenous APOBEC3B Restricts LINE-1 Retrotransposition in Transformed Cells and Human Embryonic Stem Cells*

PubMed Central

Wissing, Silke; Montano, Mauricio; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

2011-01-01

Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ∼2–3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells. PMID:21878639

Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells.

PubMed

O'Hara, M B; Hageman, J H

1990-08-01

Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process. Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent. Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. Finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

PubMed Central

Sonke, Eric; Verrydt, Megan; Postenka, Carl O.; Pardhan, Siddika; Willie, Chantalle J.; Mazzola, Clarisse R.; Hammers, Matthew D.; Pluth, Michael D.; Lobb, Ian; Power, Nicholas E.; Chambers, Ann F.; Leong, Hon S.; Sener, Alp

2016-01-01

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel–Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease. PMID:26068241

Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

PubMed

Lin, Hui-Ping; Kuo, Li-Kuo; Chuu, Chih-Pin

2012-01-01

Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients. Copyright © 2011 John Wiley & Sons, Ltd.

TPL-2 restricts Ccl24-dependent immunity to Heligmosomoides polygyrus

PubMed Central

Kannan, Yashaswini; Entwistle, Lewis J.; Pelly, Victoria S.; Perez-Lloret, Jimena; Ley, Steven C.

2017-01-01

TPL-2 (COT, MAP3K8) kinase activates the MEK1/2-ERK1/2 MAPK signaling pathway in innate immune responses following TLR, TNFR1 and IL-1R stimulation. TPL-2 contributes to type-1/Th17-mediated autoimmunity and control of intracellular pathogens. We recently demonstrated TPL-2 reduces severe airway allergy to house dust mite by negatively regulating type-2 responses. In the present study, we found that TPL-2 deficiency resulted in resistance to Heligmosomoides polygyrus infection, with accelerated worm expulsion, reduced fecal egg burden and reduced worm fitness. Using co-housing experiments, we found resistance to infection in TPL-2 deficient mice (Map3k8–/–) was independent of microbiota alterations in H. polygyrus infected WT and Map3k8–/–mice. Additionally, our data demonstrated immunity to H. polygyrus infection in TPL-2 deficient mice was not due to dysregulated type-2 immune responses. Genome-wide analysis of intestinal tissue from infected TPL-2-deficient mice identified elevated expression of genes involved in chemotaxis and homing of leukocytes and cells, including Ccl24 and alternatively activated genes. Indeed, Map3k8–/–mice had a significant influx of eosinophils, neutrophils, monocytes and Il4GFP+ T cells. Conditional knockout experiments demonstrated that specific deletion of TPL-2 in CD11c+ cells, but not Villin+ epithelial cells, LysM+ myeloid cells or CD4+ T cells, led to accelerated resistance to H. polygyrus. In line with a central role of CD11c+ cells, CD11c+ CD11b+ cells isolated from TPL-2-deficient mice had elevated Ccl24. Finally, Ccl24 neutralization in TPL-2 deficient mice significantly decreased the expression of Arg1, Retnla, Chil3 and Ear11 correlating with a loss of resistance to H. polygyrus. These observations suggest that TPL-2-regulated Ccl24 in CD11c+CD11b+ cells prevents accelerated type-2 mediated immunity to H. polygyrus. Collectively, this study identifies a previously unappreciated role for TPL-2 controlling immune responses to H. polygyrus infection by restricting Ccl24 production. PMID:28759611

Cell-mediated immunity in herpes simplex virus-infected mice: H-2 mapping of the delayed-type hypersensitivity response and the antiviral T cell response.

PubMed

Nash, A A; Phelan, J; Wildy, P

1981-04-01

An adoptive transfer system was used to investigate the H-2 restriction of delayed-type hypersensitivity (DTH) to herpes simplex virus. A successful DTH transfer was achieved when donor and recipient were compatible at the I-A region, with K and D region compatibility unnecessary. However, the rapid clearance of infectious virus from the inoculation site was found only when the donor and recipients were compatible at H-2K (and presumably D) and I-A regions.

Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets.

PubMed

van den Brand, Judith M A; Wohlsein, Peter; Herfst, Sander; Bodewes, Rogier; Pfankuche, Vanessa M; van de Bildt, Marco W G; Seehusen, Frauke; Puff, Christina; Richard, Mathilde; Siebert, Ursula; Lehnert, Kristina; Bestebroer, Theo; Lexmond, Pascal; Fouchier, Ron A M; Prenger-Berninghoff, Ellen; Herbst, Werner; Koopmans, Marion; Osterhaus, Albert D M E; Kuiken, Thijs; Baumgärtner, Wolfgang

2016-01-01

Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7 (Seal/H10N7) in harbor seals (Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.

GENERATION OF CYTOTOXIC LYMPHOCYTES IN MIXED LYMPHOCYTE REACTIONS

PubMed Central

Forman, James; Möller, Göran

1973-01-01

Generation of cytotoxic effector cells by a unidirectional mixed lymphocyte reaction (MLR) in the mouse H-2 system was studied using labeled YAC (H-2a) leukemia cells as targets. The responding effector cell displayed a specific cytotoxic effect against target cells of the same H-2 genotype as the stimulating cell population. Killing of syngeneic H-2 cells was not observed, even when the labeled target cells were "innocent bystanders" in cultures where specific target cells were reintroduced. Similar results were found with spleen cells taken from mice sensitized in vivo 7 days earlier. The effector cell was not an adherent cell and was not activated by supernatants from MLR. The supernatants were not cytotoxic by themselves. When concanavalin A or phytohemagglutinin was added to the cytotoxic test system, target and effector cells were agglutinated. Under these conditions, killing of H-2a target cells was observed in mixed cultures where H-2a lymphocytes were also the effector cells. These findings indicate that specifically activated, probably thymus-derived lymphocytes, can kill nonspecifically once they have been activated and providing there is close contact between effector and target cells. Thus, specificity of T cell killing appears to be restricted to recognition and subsequent binding to the targets, the actual effector phase being nonspecific. PMID:4269560

Identification of two novel immunodominant UreB CD4(+) T cell epitopes in Helicobacter pylori infected subjects.

PubMed

Yang, Wu-Chen; Chen, Li; Li, Hai-Bo; Li, Bin; Hu, Jian; Zhang, Jin-Yong; Yang, Shi-Ming; Zou, Quan-Ming; Guo, Hong; Wu, Chao

2013-02-06

An epitope-based vaccine is a promising option for treating Helicobacter pylori (H. pylori) infection. Epitope mapping is the first step in designing an epitope-based vaccine. A pivotal role of CD4(+) T cells in protection against H. pylori has been accepted, but few Th epitopes have been identified. In this study, two novel UreB CD4(+) T cell epitopes were identified using PBMCs obtained from two H. pylori infected subjects. We determined the restriction molecules by antibody blocking and used various Epstein-Barr virus-transformed B lymphocyte cell lines (BLCLs) with different HLA alleles as APCs to present peptides to CD4(+) T cells. These epitopes were DRB1*1404-restricted UreB(373-385) and DRB1*0803-restricted UreB(438-452). The T cells specific to these epitopes not only recognized autologous DCs loaded with recombinant UreB but also those pulsed with H. pylori whole cell lysates, suggesting that these epitope peptides are naturally processed. These epitopes have important value for designing an effective H. pylori vaccine. Copyright © 2012 Elsevier Ltd. All rights reserved.

Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection.

PubMed

Jessen, Birthe; Faller, Simone; Krempl, Christine D; Ehl, Stephan

2011-10-01

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.

Major Histocompatibility Complex-Dependent Cytotoxic T Lymphocyte Repertoire and Functional Avidity Contribute to Strain-Specific Disease Susceptibility after Murine Respiratory Syncytial Virus Infection ▿

PubMed Central

Jessen, Birthe; Faller, Simone; Krempl, Christine D.; Ehl, Stephan

2011-01-01

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2d allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2d mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2+ CTL directed against the immunodominant epitope M2-1 82, while in H-2b mice only 20% of antiviral CTL were Vβ9+ CTL specific for the immunodominant epitope M187. The immunodominant H-2d-restricted CTL lysed target cells less efficiently than the immunodominant H-2b CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2d mating (C57BL/6-H-2dxb mice) attenuated disease. Moreover, disease in H-2d mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2+ cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection. PMID:21795345

Influence of Chronic Moderate Sleep Restriction and Exercise Training on Anxiety, Spatial Memory, and Associated Neurobiological Measures in Mice

PubMed Central

Zielinski, Mark R.; Davis, J. Mark; Fadel, James R.; Youngstedt, Shawn D.

2013-01-01

Sleep deprivation can have deleterious effects on cognitive function and mental health. Moderate exercise training has myriad beneficial effects on cognition and mental health. However, physiological and behavioral effects of chronic moderate sleep restriction and its interaction with common activities, such as moderate exercise training, have received little investigation. The aims of this study were to examine the effects of chronic moderate sleep restriction and moderate exercise training on anxiety-related behavior, spatial memory, and neurobiological correlates in mice. Male mice were randomized to one of four 11-week treatments in a 2 [sleep restriction (~4 h loss/day) vs. ad libitum sleep] × 2 [exercise (1 h/day/6 d/wk) vs. sedentary activity] experimental design. Anxiety-related behavior was assessed with the elevated-plus maze, and spatial learning and memory were assessed with the Morris water maze. Chronic moderate sleep restriction did not alter anxiety-related behavior, but exercise training significantly attenuated anxiety-related behavior. Spatial learning and recall, hippocampal cell activity (i.e., number of c-Fos positive cells), and brain derived neurotrophic factor were significantly lower after chronic moderate sleep restriction, but higher after exercise training. Further, the benefit of exercise training for some memory variables was evident under normal sleep, but not chronic moderate sleep restriction conditions. These data indicate clear detrimental effects of chronic moderate sleep restriction on spatial memory and that the benefits of exercise training were impaired after chronic moderate sleep restriction. PMID:23644185

Cell-mediated T lymphocyte responses against syngeneic cells modified with amino-reactive hapten (AED-NH2): H-2Dk serves as an element for cell-mediated lympholysis to amino-reactive hapten (AED-NH2)-modified self.

PubMed

Mizuochi, T; Fujiwara, H; Takai, Y; Hamaoka, T

1985-02-01

Spleen cells from C3H/He mice immunized to the newly synthesized amino-reactive hapten, 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide (AED-NH2), were stimulated in vitro with AED-NH2 modified syngeneic cells. After 5 days of culture, effector cells were assayed for their cytotoxic activity against AED-NH2-modified target blast cells. In contrast to other amino-reactive haptens reported so far, a strong cytotoxic activity against AED-NH2-modified syngeneic cells was found in H-2b mice as well as in H-2k mice. Furthermore, Dk-restricted anti-AED-NH2 CTL recognition was observed in H-2k mice as shown by cold target inhibition. Previous studies have demonstrated the predominant influence of K over D region self determinants, and of the chemical reactivity of the haptenic reagent in Ir gene control of CTL response to hapten-self. The present report illustrates the importance of the hapten itself in genetic regulation of these CTL responses.

Identification of a conjunctivitis-associated gene locus from the virulence plasmid of Yersinia enterocolitica.

PubMed

Miliotis, M D; Morris, J G; Cianciosi, S; Wright, A C; Wood, P K; Robins-Browne, R M

1990-08-01

The virulence plasmid (pYV) of Yersinia enterocolitica is necessary for production of conjunctivitis in guinea pigs and for mouse lethality. To identify the genes responsible for production of conjunctivitis in guinea pigs, we subcloned the BamHI and SalI restriction fragments of the virulence plasmid of Y. enterocolitica A2635 (serotype O:8) into derivatives of the broad-host-range plasmid pRK290 and introduced the constructions into plasmid-negative Y. enterocolitica strains. A mild, transient conjunctivitis was evident 24 h after inoculation with strains containing a 2.8-kilobase (kb) BamHI fragment of pYV. These strains were cytotoxic to HEp-2 cells but did not cause death in iron-loaded adult mice. When the 2.8- and adjacent 0.5-kb BamHI fragments were deleted from the virulence plasmid of a fully virulent Y. enterocolitica isolate, the resultant strain did not cause conjunctivitis in guinea pigs and was not cytotoxic to HEp-2 cells. However, the strain with the deletion appeared to be more virulent for mice, with more rapid dissemination after orogastric inoculation, compared with that of the parent strain. When the deletion was complemented by introduction of a plasmid containing the 2.8-kb BamHI fragment, the strain again caused conjunctivitis but had decreased virulence for mice.

Protective effects of protopine on hydrogen peroxide-induced oxidative injury of PC12 cells via Ca(2+) antagonism and antioxidant mechanisms.

PubMed

Xiao, Xianghua; Liu, Juntian; Hu, Jingwen; Zhu, Xiuping; Yang, Hua; Wang, Chaoyun; Zhang, Yuanhui

2008-09-04

Calcium and lipid peroxidation play important roles in oxidative stress-induced cellular injury and apoptosis, which ultimately cause cell death. In this study we examined whether protopine had a neuroprotection against H(2)O(2)-induced injury in PC12 cells. Pretreatment of PC12 cells with protopine improved the cell viability, enhanced activities of superoxide dismutase, glutathione peroxidase and catalase, and decreased malondialdehyde level in the H(2)O(2) injured cells. Protopine also reversed the increased intracellular Ca(2+) concentration and the reduced mitochondrial membrane potential caused by H(2)O(2) in the cells. Furthermore, protopine was able to inhibit caspase-3 expression and cell apoptosis induced by H(2)O(2). In summary, this study demonstrates that protopine is able to relieve H(2)O(2)-induced oxidative stress and apoptosis in PC12 cells, at least in part, by Ca(2+) antagonism and antioxidant mechanisms.

Genetic stability of RSV-F expression and the restricted growth phenotype of a live attenuated PIV3 vectored RSV vaccine candidate (MEDI-534) following restrictive growth in human lung cells.

PubMed

Nelson, Christine L; Tang, Roderick S; Stillman, Elizabeth A

2013-08-12

MEDI-534 is the first live, attenuated and vectored respiratory syncytial virus (RSV) vaccine to be evaluated in seronegative children. It consists of a bovine/human parainfluenza virus type 3 (PIV3) backbone with the RSV fusion glycoprotein (RSV-F) expressed from the second position. The PIV3 fusion and hemaglutinin-neuraminidase proteins are human-derived. No small animal appropriately replicates the restrictive growth of bovine PIV3 (bPIV3) based viruses relative to human PIV3 (hPIV3) observed in the respiratory tract of rhesus monkeys and humans, making analysis of the genetic stability of the attenuation phenotype and maintenance of RSV-F expression difficult. Screening of multiple cell-lines identified MRC-5 cells as supporting permissive growth of hPIV3 while restricting bPIV3 and MEDI-534 growth. In MRC-5 cells, the peak titers of MEDI-534 were more than 20-fold lower compared to hPIV3 peak titers. After more than 10 multicycle passages in MRC-5 cells, genetic alterations were detected in MEDI-534 that contributed to a partial loss in restricted growth in MRC-5 cells and a decrease in RSV-F expression. These adaptive mutations did not occur in the RSV-F gene but were found in the polyA sequence upstream of the transgene. MRC-5 adapted MEDI-534 viruses (1) lost some attenuation but did not replicate to the level of hPIV3 in this cell line, (2) did not completely lose RSV-F expression and (3) were able to elicit a protective anti-RSV immune response in hamsters despite lower levels of RSV-F expression. Interestingly analysis of shed MEDI-534 from a recent clinical trial indicates that in some recipients similar mutations arise by day 7 or day 12 post immunization (in press) suggesting that these mutations can arise rapidly in the human host. The utility and limits of MRC-5 cells for characterizing the attenuation and RSV-F expression of MEDI-534 is discussed. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

PubMed Central

Saw, Wan Ting; Eisenberg, Roselyn J.; Cohen, Gary H.

2016-01-01

ABSTRACT Receptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH2/gL2), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD1) and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing the activity of the mutants. Under low-pH conditions or when using deregulated forms of gD1 and gH2/gL2, the fusogenic potential of gB could only be increased in the absence of receptor, underlining the exquisite regulation that occurs in the presence of receptor. Our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. PMID:27630245

Simultaneous measurement of passage through the restriction point and MCM loading in single cells

PubMed Central

Håland, T. W.; Boye, E.; Stokke, T.; Grallert, B.; Syljuåsen, R. G.

2015-01-01

Passage through the Retinoblastoma protein (RB1)-dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyper-phosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1-phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells. PMID:26250117

Caloric restriction delays yeast chronological aging by remodeling carbohydrate and lipid metabolism, altering peroxisomal and mitochondrial functionalities, and postponing the onsets of apoptotic and liponecrotic modes of regulated cell death

PubMed Central

Arlia-Ciommo, Anthony; Leonov, Anna; Beach, Adam; Richard, Vincent R.; Bourque, Simon D.; Burstein, Michelle T.; Kyryakov, Pavlo; Gomez-Perez, Alejandra; Koupaki, Olivia; Feldman, Rachel; Titorenko, Vladimir I.

2018-01-01

A dietary regimen of caloric restriction delays aging in evolutionarily distant eukaryotes, including the budding yeast Saccharomyces cerevisiae. Here, we assessed how caloric restriction influences morphological, biochemical and cell biological properties of chronologically aging yeast advancing through different stages of the aging process. Our findings revealed that this low-calorie diet slows yeast chronological aging by mechanisms that coordinate the spatiotemporal dynamics of various cellular processes before entry into a non-proliferative state and after such entry. Caloric restriction causes a stepwise establishment of an aging-delaying cellular pattern by tuning a network that assimilates the following: 1) pathways of carbohydrate and lipid metabolism; 2) communications between the endoplasmic reticulum, lipid droplets, peroxisomes, mitochondria and the cytosol; and 3) a balance between the processes of mitochondrial fusion and fission. Through different phases of the aging process, the caloric restriction-dependent remodeling of this intricate network 1) postpones the age-related onsets of apoptotic and liponecrotic modes of regulated cell death; and 2) actively increases the chance of cell survival by supporting the maintenance of cellular proteostasis. Because caloric restriction decreases the risk of cell death and actively increases the chance of cell survival throughout chronological lifespan, this dietary intervention extends longevity of chronologically aging yeast. PMID:29662634

Co-expression of HLA-B7 and HLA-B27 alleles is associated with B7-restricted immunodominant responses following influenza infection.

PubMed

Akram, Ali; Inman, Robert D

2013-12-01

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2-K(-/-)/D(-/-) double-knockout transgenic mice expressing either one or two human MHC-I alleles. We hypothesized that co-expression of different allele combinations figures critically in immunodominance and examined this in influenza-infected, double Tg MHC-I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2-restricted matrix I.58-66, the B7-restricted NP418-426 or the B27-restricted NP383-391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu-infected B7/B27 mice, a significantly reduced level of B27/NP383-restricted CTL response was detected while there was no change in the B7/NP418-restricted CTL response. Flu-specific tetramer studies revealed a partial deletion of Vβ8.1(+) NP383/B27-restricted CD8(+) T cells, and a diminished Vβ12(+) CD8(+) T-cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co-dominant expression of MHC-I alleles for host immune response to pathogens. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm.

PubMed

Richardson, Sarah L; Swietach, Pawel

2016-10-25

During capillary transit, red blood cells (RBCs) must exchange large quantities of CO 2 and O 2 in typically less than one second, but the degree to which this is rate-limited by diffusion through cytoplasm is not known. Gas diffusivity is intuitively assumed to be fast and this would imply that the intracellular path-length, defined by RBC shape, is not a factor that could meaningfully compromise physiology. Here, we evaluated CO 2 diffusivity (D CO2 ) in RBCs and related our results to cell shape. D CO2 inside RBCs was determined by fluorescence imaging of [H + ] dynamics in cells under superfusion. This method is based on the principle that H + diffusion is facilitated by CO 2 /HCO 3 - buffer and thus provides a read-out of D CO2 . By imaging the spread of H + ions from a photochemically-activated source (6-nitroveratraldehyde), D CO2 in human RBCs was calculated to be only 5% of the rate in water. Measurements on RBCs containing different hemoglobin concentrations demonstrated a halving of D CO2 with every 75 g/L increase in mean corpuscular hemoglobin concentration (MCHC). Thus, to compensate for highly-restricted cytoplasmic diffusion, RBC thickness must be reduced as appropriate for its MCHC. This can explain the inverse relationship between MCHC and RBC thickness determined from >250 animal species.

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm

PubMed Central

Richardson, Sarah L.; Swietach, Pawel

2016-01-01

During capillary transit, red blood cells (RBCs) must exchange large quantities of CO2 and O2 in typically less than one second, but the degree to which this is rate-limited by diffusion through cytoplasm is not known. Gas diffusivity is intuitively assumed to be fast and this would imply that the intracellular path-length, defined by RBC shape, is not a factor that could meaningfully compromise physiology. Here, we evaluated CO2 diffusivity (DCO2) in RBCs and related our results to cell shape. DCO2 inside RBCs was determined by fluorescence imaging of [H+] dynamics in cells under superfusion. This method is based on the principle that H+ diffusion is facilitated by CO2/HCO3− buffer and thus provides a read-out of DCO2. By imaging the spread of H+ ions from a photochemically-activated source (6-nitroveratraldehyde), DCO2 in human RBCs was calculated to be only 5% of the rate in water. Measurements on RBCs containing different hemoglobin concentrations demonstrated a halving of DCO2 with every 75 g/L increase in mean corpuscular hemoglobin concentration (MCHC). Thus, to compensate for highly-restricted cytoplasmic diffusion, RBC thickness must be reduced as appropriate for its MCHC. This can explain the inverse relationship between MCHC and RBC thickness determined from >250 animal species. PMID:27777410

Role of calreticulin in the sensitivity of myocardiac H9c2 cells to oxidative stress caused by hydrogen peroxide.

PubMed

Ihara, Yoshito; Urata, Yoshishige; Goto, Shinji; Kondo, Takahito

2006-01-01

Calreticulin (CRT), a Ca2+-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac apoptosis in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In the present study, the effect of overexpression of CRT on susceptibility to apoptosis under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. Under oxidative stress due to H2O2, the CRT-overexpressing cells were highly susceptible to apoptosis compared with controls. In the overexpressing cells, the levels of cytoplasmic free Ca2+ ([Ca2+]i) were significantly increased by H2O2, whereas in controls, only a slight increase was observed. The H2O2-induced apoptosis was enhanced by the increase in [Ca2+]i caused by thapsigargin in control cells but was suppressed by BAPTA-AM, a cell-permeable Ca2+ chelator in the CRT-overexpressing cells, indicating the importance of the level of [Ca2+]i in the sensitivity to H2O2-induced apoptosis. Suppression of CRT by the introduction of the antisense cDNA of CRT enhanced cytoprotection against oxidative stress compared with controls. Furthermore, we found that the levels of activity of calpain and caspase-12 were elevated through the regulation of [Ca2+]i in the CRT-overexpressing cells treated with H2O2 compared with controls. Thus we conclude that the level of CRT regulates the sensitivity to apoptosis under oxidative stress due to H2O2 through a change in Ca2+ homeostasis and the regulation of the Ca2+-calpain-caspase-12 pathway in myocardiac cells.

Synergistic effects of ascorbate and sorafenib in hepatocellular carcinoma: New insights into ascorbate cytotoxicity

PubMed Central

Bisetto, Sara; Newberg, Andrew; Doria, Cataldo; Levine, Mark; Monti, Daniel A.; Hoek, Jan B.

2016-01-01

We investigated the mechanism of selective ascorbate-induced cytotoxicity in tumor cells, including Hep G2 cells, compared to primary hepatocytes. H2O2 formation was required for ascorbate cytotoxicity, as extracellular catalase treatment protected tumor cells. H2O2 generated by glucose oxidase treatment also caused cell killing, but treatment with a pharmacological dose (5-20 mM) of ascorbate was significantly more cytotoxic at comparable rates of H2O2 production, suggesting that ascorbate enhanced H2O2 cytotoxicity. This was further supported by the finding that ascorbate at a non-cytotoxic dose (1 mM) enhanced cell killing caused by glucose oxidase. Consistent with this conclusion, ascorbate treatment caused deregulation of cellular calcium homeostasis, resulting in massive mitochondrial calcium accumulation. Ascorbate acted synergistically with the chemotherapeutic sorafenib in killing Hep G2 cells, but not primary hepatocytes, suggesting adjuvant ascorbate treatment can broaden sorafenib's therapeutic range. Sorafenib caused mitochondrial depolarization and prevented mitochondrial calcium sequestration. Subsequent ascorbate addition further deregulated cellular calcium homeostasis promoting cell death. Additionally, we present the case of a patient with hepatocellular carcinoma (HCC) who had prolonged regression of a rib metastasis upon combination treatment with ascorbate and sorafenib, indicating that these studies have direct clinical relevance. PMID:27036367

Synergistic effects of ascorbate and sorafenib in hepatocellular carcinoma: New insights into ascorbate cytotoxicity.

PubMed

Rouleau, Lauren; Antony, Anil Noronha; Bisetto, Sara; Newberg, Andrew; Doria, Cataldo; Levine, Mark; Monti, Daniel A; Hoek, Jan B

2016-06-01

We investigated the mechanism of selective ascorbate-induced cytotoxicity in tumor cells, including Hep G2 cells, compared to primary hepatocytes. H2O2 formation was required for ascorbate cytotoxicity, as extracellular catalase treatment protected tumor cells. H2O2 generated by glucose oxidase treatment also caused cell killing, but treatment with a pharmacologic dose (5-20mM) of ascorbate was significantly more cytotoxic at comparable rates of H2O2 production, suggesting that ascorbate enhanced H2O2 cytotoxicity. This was further supported by the finding that ascorbate at a non-cytotoxic dose (1mM) enhanced cell killing caused by glucose oxidase. Consistent with this conclusion, ascorbate treatment caused deregulation of cellular calcium homeostasis, resulting in massive mitochondrial calcium accumulation. Ascorbate acted synergistically with the chemotherapeutic sorafenib in killing Hep G2 cells, but not primary hepatocytes, suggesting adjuvant ascorbate treatment can broaden sorafenib's therapeutic range. Sorafenib caused mitochondrial depolarization and prevented mitochondrial calcium sequestration. Subsequent ascorbate addition further deregulated cellular calcium homeostasis promoting cell death. Additionally, we present the case of a patient with hepatocellular carcinoma (HCC) who had prolonged regression of a rib metastasis upon combination treatment with ascorbate and sorafenib, indicating that these studies have direct clinical relevance. Copyright © 2016 Elsevier Inc. All rights reserved.

Influence of chronic moderate sleep restriction and exercise training on anxiety, spatial memory, and associated neurobiological measures in mice.

PubMed

Zielinski, Mark R; Davis, J Mark; Fadel, James R; Youngstedt, Shawn D

2013-08-01

Sleep deprivation can have deleterious effects on cognitive function and mental health. Moderate exercise training has myriad beneficial effects on cognition and mental health. However, physiological and behavioral effects of chronic moderate sleep restriction and its interaction with common activities, such as moderate exercise training, have received little investigation. The aims of this study were to examine the effects of chronic moderate sleep restriction and moderate exercise training on anxiety-related behavior, spatial memory, and neurobiological correlates in mice. Male mice were randomized to one of four 11-week treatments in a 2 [sleep restriction (∼4h loss/day) vs. ad libitum sleep] × 2 [exercise (1h/day/6 d/wk) vs. sedentary activity] experimental design. Anxiety-related behavior was assessed with the elevated-plus maze, and spatial learning and memory were assessed with the Morris water maze. Chronic moderate sleep restriction did not alter anxiety-related behavior, but exercise training significantly attenuated anxiety-related behavior. Spatial learning and recall, hippocampal cell activity (i.e., number of c-Fos positive cells), and brain derived neurotrophic factor were significantly lower after chronic moderate sleep restriction, but higher after exercise training. Further, the benefit of exercise training for some memory variables was evident under normal sleep, but not chronic moderate sleep restriction conditions. These data indicate clear detrimental effects of chronic moderate sleep restriction on spatial memory and that the benefits of exercise training were impaired after chronic moderate sleep restriction. Published by Elsevier B.V.

Mechanical perturbation-induced ethylene releases apical dominance in Pharbitis nil by restricting shoot growth

NASA Technical Reports Server (NTRS)

Prasad, T. K.; Cline, M. G.

1985-01-01

Mechanical perturbation (MP, rubbing) or internodes of Pharbitis nil shoots initiates release of lateral buds (LB) from apical dominance within 48 h. Evidence is presented which suggests that MP promotion of LB outgrowth is mediated by ethylene-induced restriction of main shoot growth. Ethylene production in the internodes is stimulated by MP within 2 h. Effects of MP are mimicked by treatments with 1-aminocyclopropane-1-carboxylic acid (ACC) and are negated by the inhibitors of ethylene production or action, aminoethoxy vinylglycine (AVG) and AgNO3. The fact that effects of MP, ACC, and ethylene inhibitors are observed to occur on main shoot growth at least 24 h before they are observed to occur on LB growth suggests a possible cause and effect relationship. MP also causes an increase in internode diameter. MP stimulation of ethylene production appears to be mediated by ACC synthase. The results of this study and our previous studies suggest that apical dominance may be released by any mechanism which induces ethylene restriction of main shoot growth.

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells

PubMed Central

Thiel, U; Pirson, S; Müller-Spahn, C; Conrad, H; Busch, D H; Bernhard, H; Burdach, S; Richter, G H S

2011-01-01

Background: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8+ T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. Methods: Following repetitive CHM1319 (VIMPCSWWV) and EZH2666 (YMCSFLFNL) peptide-driven stimulations with HLA-A*0201+ dendritic cells (DC), allo-restricted HLA-A*0201− CD8+ T cells were stained with HLA-A*0201/peptide multimers, sorted and expanded by limiting dilution. Results: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A*0201 and killed HLA-A*0201+ ET lines expressing the antigen while HLA-A*0201– ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2−/−γC−/− mice. Within this context, we identified the CHM1319 peptide as a new candidate target antigen for ET immunotherapy. Conclusion: These results clearly identify the ET-derived antigens, EZH2666 and CHM1319, as suitable targets for protective allo-restricted human CD8+ T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation. PMID:21407224

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

PubMed Central

Stone, G. E.; Miller, O. L.; Prescott, D. M.

1965-01-01

The formation of a soluble H3-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H3-thymidine during G1 or G2. Uptake of H3-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H3-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H3-thymidine, the pool does not turn over during non-S periods. PMID:19866660

The transcription factor Olig2 is important for the biology of diffuse intrinsic pontine gliomas.

PubMed

Anderson, Jane L; Muraleedharan, Ranjithmenon; Oatman, Nicole; Klotter, Amanda; Sengupta, Satarupa; Waclaw, Ronald R; Wu, Jianqiang; Drissi, Rachid; Miles, Lili; Raabe, Eric H; Weirauch, Matthew L; Fouladi, Maryam; Chow, Lionel M; Hoffman, Lindsey; DeWire, Mariko; Dasgupta, Biplab

2017-08-01

Diffuse intrinsic pontine glioma (DIPG) is a high-grade brainstem glioma of children with dismal prognosis. There is no single unifying model about the cell of origin of DIPGs. Proliferating cells in the developing human and mouse pons, the site of DIPGs, express neural stem/progenitor cell (NPC) markers, including Sox2, nestin, vimentin, Olig2, and glial fibrillary acidic protein, in an overlapping and non-overlapping manner, suggesting progenitor cell heterogeneity in the pons. It is thought that during a restricted window of postnatal pons development, a differentiation block caused by genetic/epigenetic changes leads to unrestrained progenitor proliferation and DIPG development. Nearly 80% of DIPGs harbor a mutation in the H3F3A or the related HIST1H3B gene. Supporting the impaired differentiation model, NPCs derived from human induced pluripotent stem cells expressing the H3F3A mutation showed complete differentiation block. However, the mechanisms regulating an altered differentiation program in DIPG are unknown. We established syngeneic serum-dependent and independent primary DIPG lines, performed molecular characterization of DIPG lines in vitro and in an orthotopic xenograft model, and used small hairpin RNA to examine Olig2 function in DIPG. The transcription factor Olig2 is highly expressed in 70%-80% of DIPGs. Here we report that Olig2 expression and DIPG differentiation are mutually exclusive events in vitro, and only DIPG cells that retained Olig2 in vitro formed robust Olig2-positive brainstem glioma with 100% penetrance in a xenograft model. Our results indicate Olig2 as an onco-requisite factor in DIPG and propose investigation of Olig2 target genes as novel candidates in DIPG therapy. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

[The mechanism of phenoptosis: 2. Hayflick limit is caused by the programmed attenuation of bioenergetics].

PubMed

Trubitsin, A G

2010-01-01

This article continues earlier started theme on a substantiation of the programmed aging mechanism (phenoptosis). The concept underlying this mechanism is that the life represents a lot of the interconnected physical and chemical processes moving by the bioenergetics. The gradual programmed decrease of the level of bioenergetics causes the slow and coordinated attenuation of all physiological functions, i.e. aging. For a convincing substantiation of such mechanism it is necessary to show, how attenuation of bioenergetics causes the basic nocuous processes accompanying aging. It is shown earlier that the age dependent decrease in level of bioenergetics causes increase in production of reactive oxygen species by mitochondria and decrease in overall level of protein synthesis. The proof that Hayflick limit is also caused by the decrease in level of bioenergetics is presented in this article. Decrease in level of bioenergetics below certain critical level deprives a cell the ability to pass the restriction point of G1-phase of proliferative cycle. The inhibitor of cyclin-dependent kinase, p27, prevents the passage through this critical point in all normal cells. During division of normal somatic cells p27 is removed by cyclin E-Cdk2 complex. Interaction p27 with cyclin E-Cdk2 complex can have two consequences. At the normal physiological level of bioenergetics the cyclin E-Cdk2 phosphorylates p27, then the latter is destroyed by proteolytic enzymes--the cell enters in S-phase. When the programme decreases the bioenergetics level below certain value the cyclin E-Cdk2 becomes the target for p27. As a result the inhibitor evacuation stops and restriction point becomes closed--a cell enters irreversible proliferative rest.

Bovine Respiratory Syncytial Virus and Histophilus somni Interaction at the Alveolar Barrier

PubMed Central

Agnes, J. T.; Zekarias, B.; Shao, M.; Anderson, M. L.; Gershwin, L. J.

2013-01-01

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection. PMID:23649093

Bovine respiratory syncytial virus and Histophilus somni interaction at the alveolar barrier.

PubMed

Agnes, J T; Zekarias, B; Shao, M; Anderson, M L; Gershwin, L J; Corbeil, L B

2013-07-01

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity

USGS Publications Warehouse

Park, J.-W.; Moon, C.H.; Harmache, A.; Wargo, A.R.; Purcell, M.K.; Bremont, M.; Kurath, G.

2011-01-01

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 μm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Brüstle v. Greenpeace: Implications for Commercialisation of Translational Stem Cell Research.

PubMed

Mansnérus, Juli

2015-04-01

The lack of consensus on a common definition of the term 'embryo' has resulted in legal uncertainty affecting the permissibility of human embryonic stem cell (hESC) research and the commercialisation prospects and patenting of inventions of hESC origin in the EU. The Brüstle v. Greenpeace case, which by providing a very broad definition of a human embryo restricts the patentability of hESC-based inventions, aims at harmonising the patenting practices regarding interpretation of Article 6.2.c of Directive 98/44/ EC. It fills the gaps in national laws by providing binding interpretation guidelines for national courts. As currently no marketing authorisations have been granted to hESC-based products, implications of this judgment for translational hESC research together with other barriers to commercialisation of such research need to be analysed. In addition, whether the main obstacles relate to patenting restrictions or whether something else in the innovation system is impeding the market entry of these innovative products is discussed.

Telomere Dysfunction Induced Foci (TIF) Analysis.

PubMed

Mender, Ilgen; Shay, Jerry W

2015-11-20

Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al. , 2003). The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX, and telomeres using an antibody against one of the shelterin proteins such as TRF2 (Takai et al. , 2003; de Lange, 2002; Karlseder et al. , 1999). The method we describe here can be used in normal human and cancer cells. Other commonly used methods-Telomere Restriction Fragment (TRF) Analysis (Mender and Shay, 2015b) and Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015a)- in telomere biology can be found by clicking on the indicated links.

Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells.

PubMed

Mladinich, Megan C; Schwedes, John; Mackow, Erich R

2017-07-11

Zika virus (ZIKV) is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB) to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC) lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59) persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs), without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α) and transiently induced, but failed to secrete, IFN-β and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7), IRF9, and IFN-stimulated genes (ISGs) 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV) persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread. IMPORTANCE ZIKV persists in patients, crossing placental and neuronal barriers, damaging neurons, and causing fetal microencephaly. We found that ZIKV persistently infects brain endothelial cells that normally protect neurons from viral exposure. hBMECs are not damaged by ZIKV infection and, analogous to persistent HCV infection, ZIKV constitutively induces and evades antiviral ISG and IFN responses to continuously replicate in hBMECs. As a result, hBMECs provide a protective niche for systemic ZIKV spread and a viral reservoir localized in the normally protective blood-brain barrier. Consistent with the spread of ZIKV into neuronal compartments, ZIKV was released basolaterally from hBMECs. Our findings define hBMEC responses that contribute to persistent ZIKV infection and potential targets for clearing ZIKV infections from hBMECs. These results further suggest roles for additional ZIKV-infected ECs to facilitate viral spread and persistence in the protected placental, retinal, and testicular compartments. Copyright © 2017 Mladinich et al.

hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3.

PubMed

Søndergaard, Helle Bach; Brodin, Birger; Nielsen, Carsten Uhd

2008-06-01

The purpose of this work was to investigate the apical uptake and transepithelial transport of Gly-Sar along with the expression of the di-/tripeptide transporters hPEPT1 and hPEPT2 in human Calu-3 bronchial epithelial cells. The apical Gly-Sar uptake rate in Calu-3 cells followed Michaelis-Menten kinetics with a Km value of 1.3 +/- 0.3 mM and a Vmax value of 0.60 +/- 0.06 nmol cm(-2) min(-1). Transepithelial apical to basolateral transport of 50 microM [3H]-labelled Gly-Sar across the Calu-3 cell monolayer was pH-dependent. The Gly-Sar flux was significantly reduced in the presence of delta-aminolevulinic acid (2.5 mM), cephalexin (25 mM), and captopril (25 mM; p < 0.05, n = 3). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of both hPEPT1 and hPEPT2 mRNA in the Calu-3 cells. These findings were confirmed in healthy human bronchial cDNA. Restriction-endonuclease analysis identified hPEPT2 in Calu-3 cells to be the hPEPT2*1 haplotype. Western blotting demonstrated expression of the hPEPT1 protein (approximately 80 kDa), and the immunolabel was mainly localized in the apical membrane as judged by immunolocalization studies using confocal laser scanning microscopy (CLSM). This work presents for the first time hPEPT1 and hPEPT2*1 expression in human Calu-3 cells. Surprisingly, the results indicate that Gly-Sar uptake and transport in Calu-3 cells are hPEPT1-mediated rather than hPEPT2-mediated.

Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

PubMed

Naumenko, Ekaterina Anatolevna; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

2017-03-01

2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC 50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

IL-4-secreting eosinophils promote endometrial stromal cell proliferation and prevent Chlamydia-induced upper genital tract damage.

PubMed

Vicetti Miguel, Rodolfo D; Quispe Calla, Nirk E; Dixon, Darlene; Foster, Robert A; Gambotto, Andrea; Pavelko, Stephen D; Hall-Stoodley, Luanne; Cherpes, Thomas L

2017-08-15

Genital Chlamydia trachomatis infections in women typically are asymptomatic and do not cause permanent upper genital tract (UGT) damage. Consistent with this presentation, type 2 innate and T H 2 adaptive immune responses associated with dampened inflammation and tissue repair are elicited in the UGT of Chlamydia -infected women. Primary C. trachomatis infection of mice also causes no genital pathology, but unlike women, does not generate Chlamydia -specific T H 2 immunity. Herein, we explored the significance of type 2 innate immunity for restricting UGT tissue damage in Chlamydia -infected mice, and in initial studies intravaginally infected wild-type, IL-10 -/- , IL-4 -/- , and IL-4Rα -/- mice with low-dose C. trachomatis inoculums. Whereas Chlamydia was comparably cleared in all groups, IL-4 -/- and IL-4Rα -/- mice displayed endometrial damage not seen in wild-type or IL-10 -/- mice. Congruent with the aberrant tissue repair in mice with deficient IL-4 signaling, we found that IL-4Rα and STAT6 signaling mediated IL-4-induced endometrial stromal cell (ESC) proliferation ex vivo, and that genital administration of an IL-4-expressing adenoviral vector greatly increased in vivo ESC proliferation. Studies with IL-4-IRES-eGFP (4get) reporter mice showed eosinophils were the main IL-4-producing endometrial leukocyte (constitutively and during Chlamydia infection), whereas studies with eosinophil-deficient mice identified this innate immune cell as essential for endometrial repair during Chlamydia infection. Together, our studies reveal IL-4-producing eosinophils stimulate ESC proliferation and prevent Chlamydia -induced endometrial damage. Based on these results, it seems possible that the robust type 2 immunity elicited by Chlamydia infection of human genital tissue may analogously promote repair processes that reduce phenotypic disease expression.

Effect of long-term sleep restriction and subsequent recovery sleep on the diurnal rhythms of white blood cell subpopulations.

PubMed

Lasselin, Julie; Rehman, Javaid-Ur; Åkerstedt, Torbjorn; Lekander, Mats; Axelsson, John

2015-07-01

While acute modifications of sleep duration induces a wide array of immune function alterations, less is known of how longer periods with insufficient sleep affect immune functions and how they return to normal once recovery sleep is obtained. The purpose of the present study was to investigate the effects of five days of restricted sleep and a subsequent 7-day period of sleep recovery on white blood cell (WBC) subpopulation count and diurnal rhythms. Nine healthy males participated in a sleep protocol consisting of two baseline days (8h of sleep/night), five nights with restricted sleep (4h of sleep/night) and seven days of recovery sleep (8h of sleep/night). During nine of these days, blood was drawn hourly during night-time end every third hour during daytime, and differential WBC count was analyzed. Gradual increase across the days of sleep restriction was observed for total WBC (p<.001), monocytes (p<.001), neutrophils (p<.001) and lymphocytes (p<.05). Subsequent recovery sleep resulted in a gradual decrease in monocytes (p<.001) and lymphocytes (p=.001), but not in neutrophils that remained elevated over baseline level at the end of the 7-day recovery period. These effects were associated with altered diurnal rhythms of total WBC and neutrophils, restricted sleep being associated with higher levels during the night and at awakening, resulting in a flattening of the rhythm. The diurnal alterations were reversed when recovery sleep was allowed, although the amplitude of total WBC, neutrophils and monocytes was increased at the end of the recovery period in comparison to baseline. Altogether, these data show that long-term sleep restriction leads to a gradual increase of circulating WBC subpopulations and alterations of the respective diurnal rhythms. Although some of the effects caused by five days of restricted sleep were restored within the first days of recovery, some parameters were not back to baseline even after a period of seven recovery days. Copyright © 2014 Elsevier Inc. All rights reserved.

Intersection of calorie restriction and magnesium in the suppression of genome-destabilizing RNA–DNA hybrids

PubMed Central

Abraham, Karan J.; Chan, Janet N.Y.; Salvi, Jayesh S.; Ho, Brandon; Hall, Amanda; Vidya, Elva; Guo, Ru; Killackey, Samuel A.; Liu, Nancy; Lee, Jeffrey E.; Brown, Grant W.; Mekhail, Karim

2016-01-01

Dietary calorie restriction is a broadly acting intervention that extends the lifespan of various organisms from yeast to mammals. On another front, magnesium (Mg2+) is an essential biological metal critical to fundamental cellular processes and is commonly used as both a dietary supplement and treatment for some clinical conditions. If connections exist between calorie restriction and Mg2+ is unknown. Here, we show that Mg2+, acting alone or in response to dietary calorie restriction, allows eukaryotic cells to combat genome-destabilizing and lifespan-shortening accumulations of RNA–DNA hybrids, or R-loops. In an R-loop accumulation model of Pbp1-deficient Saccharomyces cerevisiae, magnesium ions guided by cell membrane Mg2+ transporters Alr1/2 act via Mg2+-sensitive R-loop suppressors Rnh1/201 and Pif1 to restore R-loop suppression, ribosomal DNA stability and cellular lifespan. Similarly, human cells deficient in ATXN2, the human ortholog of Pbp1, exhibit nuclear R-loop accumulations repressible by Mg2+ in a process that is dependent on the TRPM7 Mg2+ transporter and the RNaseH1 R-loop suppressor. Thus, we identify Mg2+ as a biochemical signal of beneficial calorie restriction, reveal an R-loop suppressing function for human ATXN2 and propose that practical magnesium supplementation regimens can be used to combat R-loop accumulation linked to the dysfunction of disease-linked human genes. PMID:27574117

Reversal of radiocontrast medium toxicity in human renal proximal tubular cells by white grape juice extract.

PubMed

Andreucci, Michele; Faga, Teresa; Pisani, Antonio; Sabbatini, Massimo; Russo, Domenico; Mattivi, Fulvio; De Sarro, Giovambattista; Navarra, Michele; Michael, Ashour

2015-03-05

Radiocontrast media (RCM)-induced nephrotoxicity (CIN) is a major clinical problem accounting for 12% of all hospital-acquired cases of acute kidney injury. The pathophysiology of CIN is not well understood, but direct toxic effects on renal cells have been postulated as contributing to CIN. We have investigated the effect of a white grape (Vitis vinifera) juice extract (WGJe) on human renal proximal tubular (HK-2) cells treated with the radiocontrast medium (RCM) sodium diatrizoate. WGJe caused an increase in phosphorylation of the prosurvival kinases Akt and ERK1/2 in HK-2 cells. Treatment of HK-2 cells with 75 mgI/ml sodium diatrizoate for 2.5h and then further incubation (for 27.5h) after removal of the RCM caused a drastic decrease in cell viability. However, pre-treatment with WGJe, prior to incubation with diatrizoate, dramatically improved cell viability. Analysis of key signaling molecules by Western blotting showed that diatrizoate caused a drastic decrease in phosphorylation of Akt (Ser473), FOXO1 (Thr24) and FOXO3a (Thr32) during the initial 2.5h incubation period, and WGJe pre-treatment caused a reversal of these effects. Further analysis by Western blotting of samples from HK-2 cells cultured for longer periods of time (for up to 27.5h after an initial 2.5h exposure to diatrizoate with or without WGJe pre-treatment) showed that WGJe pre-treatment caused a negative effect on phosphorylation of p38, NF-κB (Ser276) and pERK1/2 whilst having a positive effect on the phosphorylation of Akt, FOXO1/FOXO3a and maintained levels of Pim-1 kinase. WGJe may alleviate RCM toxicity through modulation of signaling molecules that are known to be involved in cell death and cell survival and its possible beneficial effects should be further investigated. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Reduction in cardiolipin decreases mitochondrial spare respiratory capacity and increases glucose transport into and across human brain cerebral microvascular endothelial cells.

PubMed

Nguyen, Hieu M; Mejia, Edgard M; Chang, Wenguang; Wang, Ying; Watson, Emily; On, Ngoc; Miller, Donald W; Hatch, Grant M

2016-10-01

Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial cardiolipin and decreased mitochondrial spare respiratory capacity. The reduced cardiolipin results in an increased activity of adenosine monophosphate kinase (pAMPK) and protein kinase B (pAKT) and decreased activity of glycogen synthase kinase 3 beta (pGSK3β) which results in elevated glucose transporter-1 (GLUT-1) expression and association with membranes. This in turn increases 2-dexoyglucose uptake from the apical medium into the cells with a resultant 2-deoxyglucose movement into the basolateral medium. © 2016 International Society for Neurochemistry.

Effects of flooring and restricted freestall access on behavior and claw health of dairy heifers.

PubMed

Ouweltjes, W; van der Werf, J T N; Frankena, K; van Leeuwen, J L

2011-02-01

Claw health, locomotion, feed intake, milk yield, body weight, activity, and lying and standing behavior of dairy heifers were monitored in a single dairy herd during the first 3 mo after calving. During the first 8 wk after calving, 2 treatments were applied: restricted freestall access by closing the stalls between 2300 h and 0500 h (yes or no) and alley flooring (concrete or rubber topped slatted floors). Apart from treatments, housing was identical. The animals were kept in small groups (n=4 to 6) in adjacent barn pens. Thereafter, the animals were kept in 1 group in a freestall section with concrete slatted floor and unrestricted access to the stalls for 5 wk. All animals were fed the same partial mixed ration. We hypothesized that (1) hard flooring causes high mechanical load of the claws and (2) restricted freestall access causes prolonged standing bouts and reinforced effects of hard flooring on claws. The heifers had only minor claw lesions before first calving, and the prevalence and severity of sole hemorrhages increased during the first 3 mo after calving (from 0.24 ± 0.08 to 1.18 ± 0.14 and from 0.04 ± 0.01 to 0.24 ± 0.02, respectively), particularly in the outer hind claws. Animals kept on rubber alley flooring had lower average hemorrhage scores in wk 9 (0.13 ± 0.03 vs. 0.21 ± 0.03) and wk 14 (0.20 ± 0.03 vs. 0.27 ± 0.03) after calving, had a slower feed intake (3.05 ± 0.14 vs. 3.46 ± 0.14 g/s) and spent more time feeding (7.3 ± 0.3 vs. 6.6 ± 0.3 min/h) than animals kept on hard concrete alley floors. Restricted freestall access resulted in fewer standing bouts per day (14.4 ± 1.0 vs. 17.9 ± 1.0) and more strides per hour (99.8 ± 5.4 vs. 87.2 ± 5.4) without changing overall standing time (15.0 ± 0.3 vs. 14.7 ± 0.3 h/d) and did not affect the occurrence of sole hemorrhages. The animals with no overnight freestall access spent more time standing (55.9 ± 0.9 vs. 35.8 ± 0.9 min/h) and feeding (7.8 ± 0.3 vs. 4.3 ± 0.3 min/h) between 2300 and 0500 h and less during the rest of the 24-h period (31.3 ± 0.8 vs. 37.0 ± 0.8 min/h and 6.8 ± 0.3 vs. 7.6 ± 0.3 min/h). Thus, the animals adapted to restricted freestall access, that caused increased overnight standing, by additional lying down during the day and used part of the extra standing time at night for feeding. The restrictions probably had only a minor effect on the mechanical load of their claws. Therefore, the first part of the hypothesis was confirmed and the second part was rejected. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma.

PubMed

Mazzocco, Marta; Martini, Matteo; Rosato, Antonio; Stefani, Elisabetta; Matucci, Andrea; Dalla Santa, Silvia; De Sanctis, Francesco; Ugel, Stefano; Sandri, Sara; Ferrarini, Giovanna; Cestari, Tiziana; Ferrari, Sergio; Zanovello, Paola; Bronte, Vincenzo; Sartoris, Silvia

2015-09-01

In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL)-mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 L(d). Increase of H-2 L(d) expression by cDNA transfection (Sp6/B7/L(d)) raised tumour immune protection and shifted most CTL responses towards H-2 L(d)-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 L(d)-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/L(d) cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost. © 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.

Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma

PubMed Central

Mazzocco, Marta; Martini, Matteo; Rosato, Antonio; Stefani, Elisabetta; Matucci, Andrea; Dalla Santa, Silvia; De Sanctis, Francesco; Ugel, Stefano; Sandri, Sara; Ferrarini, Giovanna; Cestari, Tiziana; Ferrari, Sergio; Zanovello, Paola; Bronte, Vincenzo; Sartoris, Silvia

2015-01-01

In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL) -mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 Ld. Increase of H-2 Ld expression by cDNA transfection (Sp6/B7/Ld) raised tumour immune protection and shifted most CTL responses towards H-2 Ld-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 Ld-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/Ld cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost. PMID:25959091

Inhibition of cell proliferation and migration by oxidative stress from ascorbate-driven juglone redox cycling in human bladder-derived T24 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kviecinski, M.R., E-mail: mrkviecinski@hotmail.com; Pedrosa, R.C., E-mail: rozangelapedrosa@gmail.com; Felipe, K.B., E-mail: kakabettega@yahoo.com.br

2012-05-04

Highlights: Black-Right-Pointing-Pointer The cytotoxicity of juglone is markedly increased by ascorbate. Black-Right-Pointing-Pointer T24 cell death by oxidative stress is necrosis-like. Black-Right-Pointing-Pointer Redox cycling by juglone/ascorbate inhibits cell proliferation. Black-Right-Pointing-Pointer Cellular migration is impaired by juglone/ascorbate. -- Abstract: The effects of juglone on T24 cells were assessed in the presence and absence of ascorbate. The EC{sub 50} value for juglone at 24 h decreased from 28.5 {mu}M to 6.3 {mu}M in the presence of ascorbate. In juglone-treated cells, ascorbate increased ROS formation (4-fold) and depleted GSH (65%). N-acetylcysteine or catalase restricted the juglone/ascorbate-mediated effects, highlighting the role of oxidative stress inmore » juglone cytotoxicity. Juglone alone or associated with ascorbate did not cause caspase-3 activation or PARP cleavage, suggesting necrosis-like cell death. DNA damage and the mild ER stress caused by juglone were both enhanced by ascorbate. In cells treated with juglone (1-5 {mu}M), a concentration-dependent decrease in cell proliferation was observed. Ascorbate did not impair cell proliferation but its association with juglone led to a clonogenic death state. The motility of ascorbate-treated cells was not affected. Juglone slightly restricted motility, but cells lost their ability to migrate most noticeably when treated with juglone plus ascorbate. We postulate that juglone kills cells by a necrosis-like mechanism inhibiting cell proliferation and the motility of T24 cells. These effects are enhanced in the presence of ascorbate.« less

Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor transgenic CD8+ T Cells specifically inhibit Ewing sarcoma growth in vitro and in vivo

PubMed Central

Kirschner, Andreas; Thiede, Melanie; Rubio, Rebeca Alba; Schirmer, David; Kirchner, Thomas; Richter, Gunther H.S.; Mall, Sabine; Klar, Richard; Riddell, Stanley; Busch, Dirk H.; Krackhardt, Angela; Grunewald, Thomas G.P.; Burdach, Stefan

2016-01-01

The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion. In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction. After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2−/−ɣc−/− mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells. CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic. PMID:27281613

Duck Interferon-Inducible Transmembrane Protein 3 Mediates Restriction of Influenza Viruses.

PubMed

Blyth, Graham A D; Chan, Wing Fuk; Webster, Robert G; Magor, Katharine E

2016-01-01

Interferon-inducible transmembrane proteins (IFITMs) can restrict the entry of a wide range of viruses. IFITM3 localizes to endosomes and can potently restrict the replication of influenza A viruses (IAV) and several other viruses that also enter host cells through the endocytic pathway. Here, we investigate whether IFITMs are involved in protection in ducks, the natural host of influenza virus. We identify and sequence duck IFITM1, IFITM2, IFITM3, and IFITM5. Using quantitative PCR (qPCR), we demonstrate the upregulation of these genes in lung tissue in response to highly pathogenic IAV infection by 400-fold, 30-fold, 30-fold, and 5-fold, respectively. We express each IFITM in chicken DF-1 cells and show duck IFITM1 localizes to the cell surface, while IFITM3 localizes to LAMP1-containing compartments. DF-1 cells stably expressing duck IFITM3 (but not IFITM1 or IFITM2) show increased restriction of replication of H1N1, H6N2, and H11N9 IAV strains but not vesicular stomatitis virus. Although duck and human IFITM3 share only 38% identity, critical residues for viral restriction are conserved. We generate chimeric and mutant IFITM3 proteins and show duck IFITM3 does not require its N-terminal domain for endosomal localization or antiviral function; however, this N-terminal end confers endosomal localization and antiviral function on IFITM1. In contrast to mammalian IFITM3, the conserved YXXθ endocytosis signal sequence in the N-terminal domain of duck IFITM3 is not essential for correct endosomal localization. Despite significant structural and amino acid divergence, presumably due to host-virus coevolution, duck IFITM3 is functional against IAV. Immune IFITM genes are poorly conserved across species, suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether coevolution between viruses and their natural host would result in the evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses and display few or no disease symptoms upon infection with most strains, including highly pathogenic avian influenza. We have characterized the duck IFITM locus and identified IFITM3 as an important restrictor of several influenza A viruses, including avian strains. With only 38% amino acid identity to human IFITM3, duck IFITM3 possesses antiviral function against influenza virus. Thus, despite long coevolution of virus and host effectors in the natural host, influenza virus evasion of IFITM3 restriction in ducks is not apparent. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

X-ray irradiation activates K+ channels via H2O2 signaling.

PubMed

Gibhardt, Christine S; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

2015-09-09

Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels.

X-ray irradiation activates K+ channels via H2O2 signaling

PubMed Central

Gibhardt, Christine S.; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

2015-01-01

Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

Genotoxic and cytotoxic effects of doxorubicin and silymarin on human hepatocellular carcinoma cells.

PubMed

Yurtcu, E; İşeri, Öd; Sahin, Fi

2014-12-01

The aim of this study was to investigate genotoxic and cytotoxic effects of doxorubicin, silymarin, or in combination on HepG2 cells for 24 and 48 h. Both doxorubicin and silymarin caused dose-dependent inhibition of cell proliferation. After 48 h of treatment, doxorubicin application caused dramatically increased ratio of apoptotic cells. Both 24 and 48 h of silymarin and doxorubicin-silymarin combination caused significant increases in the rate of apoptotic cells. Applications of doxorubicin and silymarin separately for 24 h led to deoxyribonucleic acid (DNA) damages. After 48 h of incubation, doxorubicin-induced genotoxic damage was 2-fold higher than the silymarin-induced damage. After 24 and 48 h, DNA damage in response to combined applications of doxorubicin and silymarin was indifferent from silymarin- and doxorubicin-induced damage respectively. There was not any difference in genotoxicity levels between incubation periods in combined applications of doxorubicin and silymarin. Lipid peroxidation levels increased in all applications. Biopharmacotherapy with chemotherapeutic agents are of interest in the issue of adjuvant therapy. Here, we demonstrate in vitro potential genotoxic and cytotoxic antitumor effect of silymarin on HepG2 cells at achievable plasma level concentrations. © The Author(s) 2014.

Life at acidic pH imposes an increased energetic cost for a eukaryotic acidophile.

PubMed

Messerli, Mark A; Amaral-Zettler, Linda A; Zettler, Erik; Jung, Sung-Kwon; Smith, Peter J S; Sogin, Mitchell L

2005-07-01

Organisms growing in acidic environments, pH<3, would be expected to possess fundamentally different molecular structures and physiological controls in comparison with similar species restricted to neutral pH. We begin to investigate this premise by determining the magnitude of the transmembrane electrochemical H+ gradient in an acidophilic Chlamydomonas sp. (ATCC PRA-125) isolated from the Rio Tinto, a heavy metal laden, acidic river (pH 1.7-2.5). This acidophile grows most rapidly at pH 2 but is capable of growth over a wide pH range (1.5-7.0), while Chlamydomonas reinhardtii is restricted to growth at pH>or=3 with optimal growth between pH 5.5 and 8.5. With the fluorescent H+ indicator, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), we show that the acidophilic Chlamydomonas maintains an average cytosolic pH of 6.6 in culture medium at both pH 2 and pH 7 while Chlamydomonas reinhardtii maintains an average cytosolic pH of 7.1 in pH 7 culture medium. The transmembrane electric potential difference of Chlamydomonas sp., measured using intracellular electrodes at both pH 2 and 7, is close to 0 mV, a rare value for plants, animals and protists. The 40,000-fold difference in [H+] could be the result of either active or passive mechanisms. Evidence for active maintenance was detected by monitoring the rate of ATP consumption. At the peak, cells consume about 7% more ATP per second in medium at pH 2 than at pH 7. This increased rate of consumption is sufficient to account for removal of H+ entering the cytosol across a membrane with relatively high permeability to H+ (7x10(-8) cm s-1). Our results indicate that the small increase in the rate of ATP consumption can account for maintenance of the transmembrane H+ gradient without the imposition of cell surface H+ barriers.

Enhanced lignin monomer production caused by cinnamic Acid and its hydroxylated derivatives inhibits soybean root growth.

PubMed

Lima, Rogério Barbosa; Salvador, Victor Hugo; dos Santos, Wanderley Dantas; Bubna, Gisele Adriana; Finger-Teixeira, Aline; Soares, Anderson Ricardo; Marchiosi, Rogério; Ferrarese, Maria de Lourdes Lucio; Ferrarese-Filho, Osvaldo

2013-01-01

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.

RESPONSES OF CELLS TO pH CHANGES IN THE MEDIUM

PubMed Central

Taylor, A. Cecil

1962-01-01

Studies were made with time-lapse motion pictures of the reactions of cells in culture to changes in their environment. The concentrations of H+, HCO3 -and CO2 in the medium were altered in such a way that each, in turn, could be maintained constant while the others were varied. Observations were made on the shape of the cells, their activity, and their relation to the substratum. Characteristic reversible changes in the cells were observed whenever environmental pH was altered. Elevation of the pH accelerated cell movements and caused contraction of the cytoplasm, while lowering of the pH retarded and eventually stopped all cell activity, causing apparent gelation of the protoplasm. These responses did not occur when HCO3 - and CO2 were varied without changing the pH. It is suggested that local pH changes in the micro-environment of a cell's surface may be a significant factor in controlling cell behavior in culture and in vivo. PMID:13993539

The effect of ethylene on root growth of Zea mays seedlings

NASA Technical Reports Server (NTRS)

Whalen, M. C.; Feldman, L. J.

1988-01-01

The control of primary root growth in Zea mays cv. Merit by ethylene was examined. At applied concentrations of ethylene equal to or greater than 0.1 microliter L-1, root elongation during 24 h was inhibited. The half-maximal response occurred at 0.6 microliter L-1 and the response saturated at 6 microliters L-1. Inhibition of elongation took place within 20 min. However, after ethylene was removed, elongation recovered to control values within 15 min. Root elongation was also inhibited by green light. The inhibition caused by a 24-h exposure to ethylene was restricted to the elongating region just behind the apex, with inhibition of cortical cell elongation being the primary contributor to the effect. Based on use of 2,5-norbornadiene, a gaseous competitive inhibitor of ethylene, it was concluded that endogenous ethylene normally inhibits root elongation.

Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection.

PubMed

Lawler, Clara; Tan, Cindy S E; Simas, J Pedro; Stevenson, Philip G

2016-10-15

Gammaherpesviruses establish persistent, systemic infections and cause cancers. Murid herpesvirus 4 (MuHV-4) provides a unique window into the early events of host colonization. It spreads via lymph nodes. While dendritic cells (DC) pass MuHV-4 to lymph node B cells, subcapsular sinus macrophages (SSM), which capture virions from the afferent lymph, restrict its spread. Understanding how this restriction works offers potential clues to a more comprehensive defense. Type I interferon (IFN-I) blocked SSM lytic infection and reduced lytic cycle-independent viral reporter gene expression. Plasmacytoid DC were not required, but neither were SSM the only source of IFN-I, as IFN-I blockade increased infection in both intact and SSM-depleted mice. NK cells restricted lytic SSM infection independently of IFN-I, and SSM-derived virions spread to the spleen only when both IFN-I responses and NK cells were lacking. Thus, multiple innate defenses allowed SSM to adsorb virions from the afferent lymph with relative impunity. Enhancing IFN-I and NK cell recruitment could potentially also restrict DC infection and thus improve infection control. Human gammaherpesviruses cause cancers by infecting B cells. However, vaccines designed to block virus binding to B cells have not stopped infection. Using a related gammaherpesvirus of mice, we have shown that B cells are infected not via cell-free virus but via infected myeloid cells. This suggests a different strategy to stop B cell infection: stop virus production by myeloid cells. Not all myeloid infection is productive. We show that subcapsular sinus macrophages, which do not pass infection to B cells, restrict gammaherpesvirus production by recruiting type I interferons and natural killer cells. Therefore, a vaccine that speeds the recruitment of these defenses might stop B cell infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

PubMed Central

Lawler, Clara; Tan, Cindy S. E.; Simas, J. Pedro

2016-01-01

ABSTRACT Gammaherpesviruses establish persistent, systemic infections and cause cancers. Murid herpesvirus 4 (MuHV-4) provides a unique window into the early events of host colonization. It spreads via lymph nodes. While dendritic cells (DC) pass MuHV-4 to lymph node B cells, subcapsular sinus macrophages (SSM), which capture virions from the afferent lymph, restrict its spread. Understanding how this restriction works offers potential clues to a more comprehensive defense. Type I interferon (IFN-I) blocked SSM lytic infection and reduced lytic cycle-independent viral reporter gene expression. Plasmacytoid DC were not required, but neither were SSM the only source of IFN-I, as IFN-I blockade increased infection in both intact and SSM-depleted mice. NK cells restricted lytic SSM infection independently of IFN-I, and SSM-derived virions spread to the spleen only when both IFN-I responses and NK cells were lacking. Thus, multiple innate defenses allowed SSM to adsorb virions from the afferent lymph with relative impunity. Enhancing IFN-I and NK cell recruitment could potentially also restrict DC infection and thus improve infection control. IMPORTANCE Human gammaherpesviruses cause cancers by infecting B cells. However, vaccines designed to block virus binding to B cells have not stopped infection. Using a related gammaherpesvirus of mice, we have shown that B cells are infected not via cell-free virus but via infected myeloid cells. This suggests a different strategy to stop B cell infection: stop virus production by myeloid cells. Not all myeloid infection is productive. We show that subcapsular sinus macrophages, which do not pass infection to B cells, restrict gammaherpesvirus production by recruiting type I interferons and natural killer cells. Therefore, a vaccine that speeds the recruitment of these defenses might stop B cell infection. PMID:27466430

The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide.

PubMed

Perry, Mark M; Tildy, Bernadett; Papi, Alberto; Casolari, Paolo; Caramori, Gaetano; Rempel, Karen Limbert; Halayko, Andrew J; Adcock, Ian; Chung, Kian Fan

2018-05-09

COPD is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease caused, in part, by the aberrant function of airway smooth muscle (ASM). We have previously demonstrated that hydrogen sulfide (H 2 S) can inhibit ASM cell proliferation and CXCL8 release, from cells isolated from non-smokers. We examined the effect of H 2 S upon ASM cells from COPD patients. ASM cells were isolated from non-smokers, smokers and patients with COPD (n = 9). Proliferation and cytokine release (IL-6 and CXCL8) of ASM was induced by FCS, and measured by bromodeoxyuridine incorporation and ELISA, respectively. Exposure of ASM to H 2 S donors inhibited FCS-induced proliferation and cytokine release, but was less effective upon COPD ASM cells compared to the non-smokers and smokers. The mRNA and protein expression of the enzymes responsible for endogenous H 2 S production (cystathionine-β-synthase [CBS] and 3-mercaptopyruvate sulphur transferase [MPST]) were inhibited by H 2 S donors. Finally, we report that exogenous H 2 S inhibited FCS-stimulated phosphorylation of ERK-1/2 and p38 mitogen activated protein kinases (MAPKs), in the non-smoker and smoker ASM cells, with little effect in COPD cells. H 2 S production provides a novel mechanism for the repression of ASM proliferation and cytokine release. The ability of COPD ASM cells to respond to H 2 S is attenuated in COPD ASM cells despite the presence of the enzymes responsible for H 2 S production.

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

PubMed

Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

2014-11-01

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

Subcloning the RBL-2H3 mucosal mast cell line reduces Ca2+ response heterogeneity at the single-cell level.

PubMed

Kuchtey, J; Fewtrell, C

1996-03-01

Ca2+ imaging experiments have revealed that for a wide variety of cell types, including RBL-2H3 mucosal mast cells, there are considerable cell-to-cell differences of the Ca2+ responses of individual cells. This heterogeneity is evident in both the shape and latency of the responses. Mast cells within a single microscopic field of view, which have experienced identical culture conditions and experimental preparation, display a wide variety of responses upon antigen stimulation. We have subcloned the RBL-2H3 mucosal mast cell line to test the hypothesis that genetic heterogeneity within the population is the cause of the Ca2+ response heterogeneity. We found that cell-to-cell variability was significantly reduced in four of five clonal lines. The response heterogeneity remaining within the clones was not an experimental artifact caused by differences in the amount of fura-2 loaded by individual cells. Factors other than genetic heterogeneity must partly account for Ca2+ response heterogeneity. It is possible that the complex shapes and variability of the Ca2+ responses are reflections of the fact that there are multiple factors underlying the Ca2-response to antigen stimulation. Small differences from cell to cell in one or more of these factors could be a cause of the remaining Ca2+ response heterogeneity.

A Subdominant CD8+ Cytotoxic T Lymphocyte (CTL) Epitope from the Plasmodium yoelii Circumsporozoite Protein Induces CTLs That Eliminate Infected Hepatocytes from Culture

PubMed Central

Franke, Eileen D.; Sette, Alessandro; Sacci, John; Southwood, Scott; Corradin, Giampietro; Hoffman, Stephen L.

2000-01-01

Previous studies indicated that the Plasmodium yoelii circumsporozoite protein (PyCSP) 57–70 region elicits T cells capable of eliminating infected hepatocytes in vitro. Herein, we report that the PyCSP58–67 sequence contains an H-2d binding motif, which binds purified Kd molecules in vitro with low affinity (3,267 nM) and encodes an H-2d-restricted cytotoxic T lymphocyte (CTL) epitope. Immunization of BALB/c mice with three doses of a multiple antigen peptide (MAP) construct containing four branches of amino acids 57 to 70 linked to a lysine-glycine core [MAP4(PyCSP57–70)] and Lipofectin as the adjuvant induced both T-cell proliferation and a peptide-specific CTL response that was PyCSP59–67 specific, H-2d restricted, and CD8+ T cell dependent. Immunization with either DNA encoding the PyCSP or irradiated sporozoites demonstrated that this CTL epitope is subdominant since it is not recognized in the context of whole CSP immunization. The biological relevance of this CTL response was underlined by the demonstration that it could mediate genetically restricted, CD8+- and nitric-oxide-dependent elimination of infected hepatocytes in vitro, as well as partial protection of BALB/c mice against sporozoite challenge. These findings indicate that subdominant epitopes with low major histocompatibility complex affinity can be used to engineer epitope-based vaccines and have implications for the selection of epitopes for subunit-based vaccines. PMID:10816491

pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

PubMed

Gerlach, Thomas; Hensen, Luca; Matrosovich, Tatyana; Bergmann, Janina; Winkler, Michael; Peteranderl, Christin; Klenk, Hans-Dieter; Weber, Friedemann; Herold, Susanne; Pöhlmann, Stefan; Matrosovich, Mikhail

2017-06-01

The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans. Copyright © 2017 American Society for Microbiology.

pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs

PubMed Central

Gerlach, Thomas; Hensen, Luca; Matrosovich, Tatyana; Bergmann, Janina; Winkler, Michael; Peteranderl, Christin; Klenk, Hans-Dieter; Herold, Susanne

2017-01-01

ABSTRACT The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans. PMID:28356532

Nutritional restriction and acid-base balance in white-tailed deer

USGS Publications Warehouse

DelGiudice, G.D.; Mech, L.D.; Seal, U.S.

1994-01-01

We examined the effect of progressive nutritional restriction on acid-base balance in seven captive, adult white-tailed deer (Odocoileus virginianus) from 4 February to 5 May 1988 in north central Minnesota (USA). Metabolic acidosis was indicated by low mean blood pH (7.25 to 7.33) in deer throughout the study. Mean urinary pH values declined (P = 0.020) from a mean (+SE) baseline of 8.3 +0.1 to 6.7 + 0.3 as restriction progressed. Acidemia and aciduria were associated with significant variations in mean blood CO2 (P = 0.006) and pO2 (P = 0.032), serum potassium (P = 0.004) concentrations, and with a significant (P = 0.104) handling date times group interaction in urinary potassium:creatinine values. Mean bicarbonate:carbonic acid ratios were consistently below 20:1 during nutritional restriction. Mean packed cell volume increased (P = 0.019) and serum total protein decreased (P = 0.001); thus there was evidence for progressive dehydration and net protein catabolism, respectively. Blood pCO2, serum sodium, and urinary sodium:creatinine were stable throughout the study. We propose that acidosis and aciduria are metabolic complications associated with nutritional restriction of white-tailed deer.

Activity-based anorexia is associated with reduced hippocampal cell proliferation in adolescent female rats.

PubMed

Barbarich-Marsteller, Nicole C; Fornal, Casimir A; Takase, Luiz F; Bocarsly, Miriam E; Arner, Candice; Walsh, B Timothy; Hoebel, Bartley G; Jacobs, Barry L

2013-01-01

Activity-based anorexia (ABA) is an animal model of anorexia nervosa that mimics core features of the clinical psychiatric disorder, including severe food restriction, weight loss, and hyperactivity. The ABA model is currently being used to study starvation-induced changes in the brain. Here, we examined hippocampal cell proliferation in animals with ABA (or the appropriate control conditions). Adolescent female Sprague-Dawley rats were assigned to 4 groups: control (24h/day food access), food-restricted (1h/day food access), exercise (24h/day food and wheel access), and ABA (1h/day food access, 24h/day wheel access). After 3 days of ABA, 5-bromo-2'-deoxyuridine (BrdU; 200mg/kg, i.p.) was injected and the rats were perfused 2h later. Brains were removed and subsequently processed for BrdU and Ki67 immunohistochemistry. The acute induction of ABA reduced cell proliferation in the dentate gyrus. This effect was significant in the hilus region of the dentate gyrus, but not in the subgranular zone, where adult neurogenesis occurs. Marked decreases in cell proliferation were also observed in the surrounding dorsal hippocampus and in the corpus callosum. These results indicate a primary effect on gliogenesis rather than neurogenesis following 3 days of ABA. For each brain region studied (except SGZ), there was a strong positive correlation between the level of cell proliferation and body weight/food intake. Future studies should examine whether these changes are maintained following long-term weight restoration and whether alterations in neurogenesis occur following longer exposures to ABA. Copyright © 2012 Elsevier B.V. All rights reserved.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

PubMed Central

Nicholls, Sarah; Piper, Karen P.; Mohammed, Fiyaz; Dafforn, Timothy R.; Tenzer, Stefan; Salim, Mahboob; Mahendra, Premini; Craddock, Charles; van Endert, Peter; Schild, Hansjörg; Cobbold, Mark; Engelhard, Victor H.; Moss, Paul A. H.; Willcox, Benjamin E.

2009-01-01

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition. PMID:19234124

Efficacious delivery of protein drugs to prostate cancer cells by PSMA-targeted pH-responsive chimaeric polymersomes.

PubMed

Li, Xiang; Yang, Weijing; Zou, Yan; Meng, Fenghua; Deng, Chao; Zhong, Zhiyuan

2015-12-28

Protein drugs as one of the most potent biotherapeutics have a tremendous potential in cancer therapy. Their application is, nevertheless, restricted by absence of efficacious, biocompatible, and cancer-targeting nanosystems. In this paper, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins into prostate cancer cells. Acupa-CPs had a unimodal distribution with average sizes ranging from 157-175 nm depending on amounts of Acupa. They displayed highly efficient loading of both model proteins, bovine serum albumin (BSA) and cytochrome C (CC), affording high protein loading contents of 9.1-24.5 wt.%. The in vitro release results showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane. CLSM and MTT studies demonstrated that CC-loaded Acupa10-CPs mediated efficient delivery of protein drugs into PSMA positive LNCaP cells leading to pronounced antitumor effect, in contrast to their non-targeting counterparts and free CC. Remarkably, granzyme B (GrB)-loaded Acupa10-CPs caused effective apoptosis of LNCaP cells with a low half-maximal inhibitory concentration (IC50) of 1.6 nM. Flow cytometry and CLSM studies using MitoCapture™ revealed obvious depletion of mitochondria membrane potential in LNCaP cells treated with GrB-loaded Acupa10-CPs. The preliminary in vivo experiments showed that Acupa-CPs had a long circulation time with an elimination phase half-life of 3.3h in nude mice. PSMA-targeted, pH-responsive, and chimaeric polymersomes have appeared as efficient protein nanocarriers for targeted prostate cancer therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Nuclear Exclusion of the HIV-1 host defense factor APOBEC3G requires a novel cytoplasmic retention signal and is not dependent on RNA binding.

PubMed

Bennett, Ryan P; Presnyak, Vladimir; Wedekind, Joseph E; Smith, Harold C

2008-03-21

Human APOBEC3G (hA3G) is a host factor that defends against HIV-1 as well as other exogenous retroviruses and endogenous retroelements. To this end, hA3G is restricted to the cytoplasm of T lymphocytes where it interacts with viral RNA and proteins to assemble with viral particles causing a post-entry block during reverse transcription. hA3G also exhibits a mechanism to inhibit the reverse transcription of retroelements by RNA binding and sequestration into mRNA processing centers in the cytoplasm. We have determined that the molecular basis for this specialized property of hA3G is a novel cytoplasmic retention signal (CRS) that is necessary and sufficient to restrict wild-type hA3G and chimeric constructs to the cytoplasm. The CRS resides within amino acids 113-128 and is embedded within a basic flanking sequence and does not require RNA binding to retain hA3G in the cytoplasm. Paralogs of hA3G that have nuclear or cytoplasmic distributions differ from hA3G within the region encompassing the CRS motif with respect to charge and amino acid composition. We propose that the CRS enables hA3G to interact with cytoplasmic factors, and thereby enables hA3G to serve in host cell defense by restricting an antiviral sentinel to the cytoplasm. The CRS lies in a region involved in both Gag and Vif interactions; therefore, identification of this motif has important implications for the design of therapeutics that target HIV-1 while maintaining antiviral and cellular functions.

[Embryonic stem cells - a scientific by-product of the assisted reproduction technology?].

PubMed

Sterthaus, Oliver; Zhang, Hong; De Geyter, Christian

2009-12-01

The differentiation potential of embryonic stem (ES) cells seems to be higher when compared to adult stem cells, which mainly differentiate into certain tissue types only. ES cells have the potential to play an important role in regenerative medicine as demonstrated with murine ES cells. However, with human embryonic stem cells (hESC) several obstacles still have to be overcome, when these are to be used in clinical applications. The expansion of hESC, safety issues as well as the immune-tolerance after transplantation are all problems that still have to be solved. Since 2005 the derivation of hESC lines from super-numerous embryos has become permitted in Switzerland, albeit under strictly restrictive guidelines. In 2008 the Basler hESC laboratory was successful in derivating the first hESC line with a normal chromosome complement in Switzerland (CHES2). Now, new applications allow the personalized establishment of immune-tolerant stem cells, which lead to the replacement of therapeutic cloning by induced pluripotent stem cells (iPS).

Food restriction attenuates oxidative stress in brown adipose tissue of striped hamsters acclimated to a warm temperature.

PubMed

Zhang, Ji-Ying; Zhao, Xiao-Ya; Wang, Gui-Ying; Wang, Chun-Ming; Zhao, Zhi-Jun

2016-05-01

It has been suggested that the up-regulation of uncoupling proteins (UCPs) decreases reactive oxygen species (ROS) production, in which case there should be a negative relationship between UCPs expression and ROS levels. In this study, the effects of temperature and food restriction on ROS levels and metabolic rate, UCP1 mRNA expression and antioxidant levels were examined in the brown adipose tissue (BAT) of the striped hamsters (Cricetulus barabensis). The metabolic rate and food intake of hamsters which had been restricted to 80% of ad libitum food intake, and acclimated to a warm temperature (30°C), decreased significantly compared to a control group. Hydrogen peroxide (H2O2) levels were 42.9% lower in food restricted hamsters than in the control. Malonadialdehyde (MDA) levels of hamsters acclimated to 30°C that were fed ad libitum were significantly higher than those of the control group, but 60.1% lower than hamsters that had been acclimated to the same temperature but subject to food restriction. There were significantly positive correlations between H2O2 and, MDA levels, catalase activity, and total antioxidant capacity. Cytochrome c oxidase activity and UCP1 mRNA expression significantly decreased in food restricted hamsters compared to the control. These results suggest that warmer temperatures increase oxidative stress in BAT by causing the down-regulation of UCP1 expression and decreased antioxidant activity, but food restriction may attenuate the effects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

PubMed Central

Seyed, Negar; Taheri, Tahereh; Vauchy, Charline; Dosset, Magalie; Godet, Yann; Eslamifar, Ali; Sharifi, Iraj; Adotevi, Olivier; Borg, Christophe; Rohrlich, Pierre Simon; Rafati, Sima

2014-01-01

Background There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II. Methods and Findings HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(51Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential. Conclusions Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model. PMID:25310094

Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process

PubMed Central

1985-01-01

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH- sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)- carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH- /HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule. PMID:2999293

[Effects of interferon-gamma on cytotoxicity of murine activated macrophages against murine glioma cells].

PubMed

Ohyama, K; Kikuchi, H; Oda, Y; Moritake, K; Yamasaki, T

1993-06-01

We studied the effects of mouse IFN-gamma on the cytotoxic activity of murine activated macrophages (M phi) against mouse VM-Glioma cells (H-2b). Activated M phi were obtained from peritoneal exudate cells of mice from four strains, C57BL/6 (H-2b), C3H/He(H-2k), DBA/2 (H-2d), and BALB/c (H-2d), following intraperitoneal injection of (1) LPS 200 micrograms, (2) BCG 200 micrograms, (3) C. parvum 200 micrograms, or (4) MDP 350 micrograms 7 days prior to 20-hr 51Cr release-assay. Of the various combination of mouse strains and activating agents tested, that of activated M phi of the C3H/He mouse with induction by LPS had the most tumoricidal effect against the glioma cells, which was not MHC restricted. Although LPS-activated M phi underwent marked loss of cytotoxicity following initiation of in vitro culture, this 24 hr pretreatment with IFN-gamma inhibited this reduction in tumoricidal effects in a dose-dependent fashion. On the other hand, 24 hr pretreatment of target cells with IFN-gamma did not increase their susceptibility to lysis by activated M phi. These findings suggest that IFN-gamma augments the in vitro tumoricidal activation of M phi; This effect appears to be unrelated to any influence of IFN-gamma on target sensitivity to lysis by macrophages.

In silico analysis and in vitro evaluation of immunogenic and immunomodulatory properties of promiscuous peptides derived from Leishmania infantum eukaryotic initiation factor.

PubMed

Koutsoni, Olga S; Routsias, John G; Kyriazis, Ioannis D; Barhoumi, Mourad; Guizani, Ikram; Tsakris, Athanassios; Dotsika, Eleni

2017-11-01

It is generally considered as imperative the ability to control leishmaniasis through the development of a protective vaccine capable of inducing long-lasting and protective cell-mediated immune responses. In this current study, we demonstrated potential epitopes that bind to H2 MHC class I and II molecules by conducting the in silico analysis of Leishmania infantum eukaryotic Initiation Factor (LieIF) protein, using online available algorithms. Moreover, we synthesized five peptides (16-18 amino acids long) which are part of the N-terminal portion of LieIF and contain promising MHC class I and II-restricted epitopes and afterwards, their predicted immunogenicity was evaluated in vitro by monitoring peptide-specific T-cell responses. Additionally, the immunomodulatory properties of these peptides were investigated in vitro by exploring their potential of inducing phenotypic maturation and functional differentiation of murine Bone-Marrow derived Dendritic Cells (BM-DCs). It was revealed by our data that all the synthetic peptides predicted for H2 alleles; present the property of immunogenicity. Among the synthetic peptides which contained T-cell epitopes, the peptide 52-68 aa (LieIF_2) exhibited immunomodulatory properties with the larger potential. LieIF_2-pulsed BM-DCs up-regulated the expression of the co-stimulatory surface molecules CD80 and CD86, as well as the production of the proinflammatory cytokine TNF-α and of the Th1-polarizing cytokines IL-12 and IFN-γ. The aforementioned data suggest that selected parts of LieIF could be used to develop innovative subunit protective vaccines able to induce effective immunity mediated by MHC class I-restricted as well as class II-restricted T-cell responses. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Nitric oxide activates superoxide dismutase and ascorbate peroxidase to repress the cell death induced by wounding.

PubMed

Lin, Chih-Ching; Jih, Pei-Ju; Lin, Hsin-Hung; Lin, Jeng-Shane; Chang, Ling-Lan; Shen, Yu-Hsing; Jeng, Shih-Tong

2011-10-01

Wounding caused by rain, wind, and pathogen may lead plants to onset defense response. Previous studies indicated that mechanical wounding stimulates plants to generate nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). In this study, the functions of NO and H(2)O(2) after wounding in sweet potato (Ipomoea batatas cv. Tainung 57) was further analyzed. Mechanical wounding damaged cells and resulted in necrosis, but the presence of NO donors or NO scavenger might reduce or enhance the cell death caused by wounding, respectively. The amount of H(2)O(2) induced by wounding was also decreased or increased when plants were incubated with NO donors or NO scavenger, individually. These results indicate that NO may regulate H(2)O(2) generation to affect cell death. NO-induced proteins isolated from two-dimensional electrophoresis were identified to be Copper/Zinc superoxide dismutases (CuZnSODs). The activities of CuZnSODs and ascorbate peroxidase (APX) could be enhanced by NO. In addition, the expression of CuZnSOD and APX was induced by wounding via NO, and their expression was further stimulated by NO through the generation of cGMP. The influx of calcium ions and the activity of NADPH oxidase were also involved in the NO signal transduction pathway inducing APX expression. Collectively, the generation of H(2)O(2) in wounded plants might trigger cell death. Meanwhile, the production of NO induced by wounding stimulated signal transducers including cGMP, calcium ions, and H(2)O(2) to activate CuZnSOD and APX, which further decreased H(2)O(2) level and reduced the cell death caused by wounding.

Involvement of tumour necrosis factor-α-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells

PubMed Central

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-01-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257–264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand–tumour necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses. PMID:12100718

Involvement of tumour necrosis factor-alpha-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells.

PubMed

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-07-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257-264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand-tumour necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.

Intrathymic lymphopoiesis: stromal cell-associated proliferation of T cells is independent of lymphocyte genotype.

PubMed

Kyewski, B A; Travis, M; Kaplan, H S

1984-09-01

We analyzed the genetic restriction of direct cell-cell interactions between thymocytes and a) cortical epithelial cells, b) macrophages, and c) medullary dendritic cells in the mouse thymus. Thymectomized (C3H X C57BL/Ka)F1 hybrid mice were doubly grafted with P1 and P2 neonatal thymus grafts, were lethally irradiated, and were reconstituted with a mixture of P1 and P2 bone marrow cells which differed in the Thy-1 locus. The contributions of both parental inocula to the composition of the free and stromal cell-associated T cell compartments were analyzed separately in thymic grafts of each parental strain. The lymphoid composition in both compartments essentially reflected the peripheral T cell-chimerism in the host. The development of lymphostromal complexes was not restricted by the genotype of the partner cells. Statistical analysis of the distributions of P1 and P2 T cells among free thymocytes and within individual lymphostromal complexes, however, suggests that the T cells of an individual complex are the progeny of oligoclonal proliferation. Thus, both epithelial cells and bone marrow-derived stromal cells seem to be involved in different stages of intrathymic lymphopoiesis.

Tpbpa-Cre-mediated deletion of TFAP2C leads to deregulation of Cdkn1a, Akt1 and the ERK pathway, causing placental growth arrest.

PubMed

Sharma, Neha; Kubaczka, Caroline; Kaiser, Stephanie; Nettersheim, Daniel; Mughal, Sadaf S; Riesenberg, Stefanie; Hölzel, Michael; Winterhager, Elke; Schorle, Hubert

2016-03-01

Loss of TFAP2C in mouse leads to developmental defects in the extra-embryonic compartment with lethality at embryonic day (E)7.5. To investigate the requirement of TFAP2C in later placental development, deletion of TFAP2C was induced throughout extra-embryonic ectoderm at E6.5, leading to severe placental abnormalities caused by reduced trophoblast population and resulting in embryonic retardation by E8.5. Deletion of TFAP2C in TPBPA(+) progenitors at E8.5 results in growth arrest of the junctional zone. TFAP2C regulates its target genes Cdkn1a (previously p21) and Dusp6, which are involved in repression of MAPK signaling. Loss of TFAP2C reduces activation of ERK1/2 in the placenta. Downregulation of Akt1 and reduced activation of phosphorylated AKT in the mutant placenta are accompanied by impaired glycogen synthesis. Loss of TFAP2C led to upregulation of imprinted gene H19 and downregulation of Slc38a4 and Ascl2. The placental insufficiency post E16.5 causes fetal growth restriction, with 19% lighter mutant pups. Knockdown of TFAP2C in human trophoblast choriocarcinoma JAr cells inhibited MAPK and AKT signaling. Thus, we present a model where TFAP2C in trophoblasts controls proliferation by repressing Cdkn1a and activating the MAPK pathway, further supporting differentiation of glycogen cells by activating the AKT pathway. © 2016. Published by The Company of Biologists Ltd.

Russell body gastritis with Dutcher bodies evaluated using magnification endoscopy

PubMed Central

Yorita, Kenji; Iwasaki, Takehiro; Uchita, Kunihisa; Kuroda, Naoto; Kojima, Koji; Iwamura, Shinichi; Tsutsumi, Yutaka; Ohno, Akinobu; Kataoka, Hiroaki

2017-01-01

Russell body gastritis (RBG) is an unusual type of chronic gastritis characterized by marked infiltration of Mott cells, which are plasma cells filled with spherical eosinophilic bodies referred to as Russell bodies. It was initially thought that Helicobacter pylori (H. pylori) infection was a major cause of RBG and that the infiltrating Mott cells were polyphenotypic; however, a number of cases of RBG without H. pylori infection or with monoclonal Mott cells have been reported. Thus, diagnostic difficulty exists in distinguishing RBG with monoclonal Mott cells from malignant lymphoma. Here, we report an unusual case of an 86-year-old-Japanese man with H. pylori-positive RBG. During the examination of melena, endoscopic evaluation confirmed a 13-mm whitish, flat lesion in the gastric antrum. Magnification endoscopy with narrow-band imaging suggested that the lesion was most likely a poorly differentiated adenocarcinoma. Biopsy findings were consistent with chronic gastritis with many Mott cells with intranuclear inclusions referred to as Dutcher bodies. Endoscopic submucosal dissection confirmed the diagnosis of RBG with kappa-restricted monoclonal Mott cells. Malignant lymphoma was unlikely given the paucity of cytological atypia and Ki-67 immunoreactivity of monoclonal Mott cells. This is the first reported case of RBG with endoscopic diagnosis of malignant tumor and the presence of Dutcher bodies. PMID:28874963

Hemolysis and cytotoxicity mechanisms of biodegradable magnesium and its alloys.

PubMed

Zhen, Zhen; Liu, Xiaoli; Huang, Tao; Xi, TingFei; Zheng, Yufeng

2015-01-01

Good hemocompatibility and cell compatibility are essential requirements for coronary stents, especially for biodegradable magnesium alloy stents, which could change the in situ environment after implanted. In this work, the effects of magnesium ion concentration and pH value on the hemolysis and cytotoxicity have been evaluated. Solution with different Mg(2+) concentration gradients and pH values of normal saline and cell culture media DMEM adjusted by MgCl2 and NaOH respectively were tested for the hemolysis and cell viability. Results show that even when the concentration of Mg(2+) reaches 1000 μg/mL, it has little destructive effect on erythrocyte, and the high pH value over 11 caused by the degradation is the real reason for the high hemolysis ratio. Low concentrations of Mg(2+) (<100 μg/mL) cause no cytotoxicity to L929 cells, of which the cell viability is above 80%, while high concentrations of Mg(2+) (>300 μg/mL) could induce obvious death of the L929 cells. The pH of the extract plays a synergetic effect on cytotoxicity, due to the buffer action of the cell culture medium. To validate this conclusion, commercial pure Mg using normal saline and PBS as extract was tested with the measurement of pH and Mg(2+) concentration. Pure Mg leads to a higher hemolysis ratio in normal saline (47.76%) than in buffered solution (4.38%) with different pH values and low concentration of Mg(2+). The Mg extract culture media caused no cytotoxicity, with pH=8.44 and 47.80 μg/mL Mg(2+). It is suggested that buffered solution and dynamic condition should be adopted in the hemolysis evaluation. Copyright © 2014. Published by Elsevier B.V.

Involvement of Aif1 in apoptosis triggered by lack of Hxk2 in the yeast Saccharomyces cerevisiae.

PubMed

Amigoni, Loredana; Frigerio, Gianluca; Martegani, Enzo; Colombo, Sonia

2016-05-01

We recently showed that in hxk2Δ cells, showing constitutive localization of active Ras at the mitochondria, addition of acetic acid caused an increase of both apoptotic and necrotic cells compared with the wild-type strain, providing a new role for hexokinase 2 (EC 2.7.1.1) as an anti-apoptotic factor, besides its known role as a glycolytic enzyme and as a regulator of gene transcription of several Mig1-regulated genes. We also demonstrated that apoptosis induced by lack of Hxk2 may not require the activation of Yca1. Here, we show that deletion of HXK2 causes hypersensitivity to H2O2 and that addition of this well-known apoptotic stimulus to hxk2Δ cells causes an increase in the level ROS, apoptosis and mitochondrial membrane potential. We also show that deletion of AIF1 in hxk2Δ cells enhances survival after induction of apoptosis with both H2O2 and acetic acid, rescues the reduction of both growth rate and cell size, abrogates both H2O2 and acetic acid-induced ROS accumulation and decreases cell death, suggesting that Aif1 might be involved in both H2O2 and acetic acid-induced cell death in hxk2Δ cells. Moreover, we show that active Ras proteins relocalize to the plasma membrane and to the nucleus in hxk2Δ aif1Δ cells. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

LIM kinase function and renal growth: Potential role for LIM kinases in fetal programming of kidney development.

PubMed

Sparrow, Alexander J; Sweetman, Dylan; Welham, Simon J M

2017-10-01

Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling. Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; P<0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss. Copyright © 2017 Elsevier Inc. All rights reserved.

BST-2 restricts IAV release and is countered by the viral M2 protein.

PubMed

Hu, Siqi; Yin, Lijuan; Mei, Shan; Li, Jian; Xu, Fengwen; Sun, Hong; Liu, Xiaoman; Cen, Shan; Liang, Chen; Li, Ailing; Guo, Fei

2017-02-20

BST-2 (tetherin, CD317, and HM1.24) is induced by interferon and restricts virus release by tethering the enveloped viruses to the cell surface. The effect of BST-2 on influenza A virus (IAV) infection has been inconclusive. In the present study, we report that BST-2 diminishes the production of IAV virus-like particles (VLPs) that are generated by viral neuraminidase and hemagglutinin proteins to a much greater degree than it inhibits the production of wild-type IAV particles. This relatively weaker inhibition of IAV is associated with reduction in BST-2 levels, which is caused by the M2 protein that interacts with BST-2 and leads to down-regulation of cell surface BST-2 via the proteasomal pathway. Similarly to the viral antagonist Vpu, M2 also rescues the production of human immunodeficiency virus-1 VLPs and IAV VLPs in the presence of BST-2. Replication of wild-type and the M2-deleted viruses were both inhibited by BST-2, with the M2-deleted IAV being more restricted. These data reveal one mechanism that IAV employs to counter restriction by BST-2. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

H5N1 Influenza A Virus PB1-F2 Relieves HAX-1-Mediated Restriction of Avian Virus Polymerase PA in Human Lung Cells.

PubMed

Mazel-Sanchez, B; Boal-Carvalho, I; Silva, F; Dijkman, R; Schmolke, M

2018-06-01

Highly pathogenic influenza A viruses (IAV) from avian hosts were first reported to directly infect humans 20 years ago. However, such infections are rare events, and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This short (90-amino-acid) peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1-associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammal-adapted H1N1 IAV is resistant to HAX-1 imposed restriction, while the PA of an avian-origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian-origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2-deficient H5N1 IAV is attenuated in the presence of large amounts of HAX-1. Mammal-adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in the presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans. IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species, including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear what restricts these interspecies jumps on the host side or what promotes them on the virus side. Here we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction factor of the viral polymerase complex HAX-1. Interestingly, we found that human influenza A virus polymerase complexes are already adapted to HAX-1 and do not require this function of PB1-F2. We thus propose that a functional full-length PB1-F2 supports direct transmission of bird viruses into humans. Copyright © 2018 Mazel-Sanchez et al.

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

PubMed

Keller, K M; Pollard, E C

1977-05-01

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

KDR (VEGFR2) identifies a conserved human and murine hepatic progenitor and instructs early liver development

PubMed Central

Goldman, Orit; Han, Songyan; Sourrisseau, Marion; Dziedzic, Noelle; Hamou, Wissam; Corneo, Barbara; D’Souza, Sunita; Sato, Thomas; Kotton, Darrell N.; Bissig, Karl-Dimiter; Kalir, Tamara; Jacobs, Adam; Evans, Todd; Evans, Matthew J.; Gouon-Evans, Valerie

2013-01-01

SUMMARY Understanding the fetal hepatic niche is essential for optimizing the generation of functional hepatocyte-like (hepatic) cells from human embryonic stem cells (hESCs). Here, we show that KDR (VEGFR2), previously assumed to be mostly restricted to mesodermal lineages, marks a hESC-derived hepatic progenitor. hESC-derived endoderm cells do not express KDR, but when cultured in media supporting hepatic differentiation, generate KDR+ hepatic progenitors and KDR- hepatic cells. KDR+ progenitors require active KDR signaling both to instruct their own differentiation into hepatic cells, and to support non-cell-autonomously the functional maturation of co-cultured KDR- hepatic cells. Analysis of human fetal livers suggests that similar progenitors are present in human livers. Lineage tracing in mice provides in vivo evidence of a KDR+ hepatic progenitor for fetal hepatoblasts and subsequently adult hepatocytes and cholangiocytes. Altogether, our findings reveal that KDR is a conserved marker for endoderm-derived hepatic progenitors, and a functional receptor instructing early liver development. PMID:23746980

Excess processing of oxidative damaged bases causes hypersensitivity to oxidative stress and low dose rate irradiation.

PubMed

Yoshikawa, Y; Yamasaki, A; Takatori, K; Suzuki, M; Kobayashi, J; Takao, M; Zhang-Akiyama, Q-M

2015-10-01

Ionizing radiations such as X-ray and γ-ray can directly or indirectly produce clustered or multiple damages in DNA. Previous studies have reported that overexpression of DNA glycosylases in Escherichia coli (E. coli) and human lymphoblast cells caused increased sensitivity to γ-ray and X-ray irradiation. However, the effects and the mechanisms of other radiation, such as low dose rate radiation, heavy-ion beams, or hydrogen peroxide (H2O2), are still poorly understood. In the present study, we constructed a stable HeLaS3 cell line overexpressing human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) protein. We determined the survival of HeLaS3 and HeLaS3/hOGG1 cells exposed to UV, heavy-ion beams, γ-rays, and H2O2. The results showed that HeLaS3 cells overexpressing hOGG1 were more sensitive to γ-rays, OH(•), and H2O2, but not to UV or heavy-ion beams, than control HeLaS3. We further determined the levels of 8-oxoG foci and of chromosomal double-strand breaks (DSBs) by detecting γ-H2AX foci formation in DNA. The results demonstrated that both γ-rays and H2O2 induced 8-oxoguanine (8-oxoG) foci formation in HeLaS3 cells. hOGG1-overexpressing cells had increased amounts of γ-H2AX foci and decreased amounts of 8-oxoG foci compared with HeLaS3 control cells. These results suggest that excess hOGG1 removes the oxidatively damaged 8-oxoG in DNA more efficiently and therefore generates more DSBs. Micronucleus formation also supported this conclusion. Low dose-rate γ-ray effects were also investigated. We first found that overexpression of hOGG1 also caused increased sensitivity to low dose rate γ-ray irradiation. The rate of micronucleus formation supported the notion that low dose rate irradiation increased genome instability.

Comparative analysis of the interaction of HSPs in dendritic cells, macrophages, RGM-1 cells infected by Helicobacter pylori

PubMed Central

Yao, Yongliang; Wu, Jianhong; Gu, Tao; Cheng, Yang; Li, Guangxin

2016-01-01

Helicobacter pylori may cause chronic gastritis, even gastric cancer, however, antigen-presenting cells (APCs) are most important immune cells involved in the induction and expression of the underlying inflammatory responses to resist H. pylori. To study the interaction of HSPs in dendritic cells (DCs), macrophages and RGM-1 cells infected with H. pylori, HSP-27, HSP-60, HSP-70 and HSP-90 proteins were analyzed in the mucosa tissue or serum of gastritis patients caused by H. pylori, and in cell supernatant of DCs, macrophages, RGM-1 infected by H. pylori, or in above host cells. We found that HSP-27, HSP-60, HSP-70 and HSP-90 decreased in gastric epithelial cells, but increased significantly in DCs, macrophages. Meanwhile, inflammation associated proteins iNOS-2 and COX-2 were participated in the expression of HSPs in the process of host cells defensing against H. pylori infection. These findings contribute to understand the functions of HSP-27, HSP-60, HSP-70 and HSP-90 in H. pylori infection APCs and gastric epithelial cells indicating that HSPs would be diagnostic markers for H. pylori infection. PMID:27830002

CD147/EMMPRIN acts as a functional entry receptor for measles virus on epithelial cells.

PubMed

Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Terao-Muto, Yuri; Sato, Hiroki; Kai, Chieko

2010-05-01

Measles is a highly contagious human disease caused by measles virus (MeV) and remains the leading cause of death in children, particularly in developing countries. Wild-type MeV preferentially infects lymphocytes by using signaling lymphocytic activation molecule (SLAM), whose expression is restricted to hematopoietic cells, as a receptor. MeV also infects other epithelial and neuronal cells that do not express SLAM and causes pneumonia and diarrhea and, sometimes, serious symptoms such as measles encephalitis and subacute sclerosing panencephalitis. The discrepancy between the tissue tropism of MeV and the distribution of SLAM-positive cells suggests that there are unknown receptors other than SLAM for MeV. Here we identified CD147/EMMPRIN (extracellular matrix metalloproteinase inducer), a transmembrane glycoprotein, which acts as a receptor for MeV on epithelial cells. Furthermore, we found the incorporation of cyclophilin B (CypB), a cellular ligand for CD147, in MeV virions, and showed that inhibition of CypB incorporation significantly attenuated SLAM-independent infection on epithelial cells, while it had no effect on SLAM-dependent infection. To date, MeV infection was considered to be triggered by binding of its hemagglutinin (H) protein and cellular receptors. Our present study, however, indicates that MeV infection also occurs via CD147 and virion-associated CypB, independently of MeV H. Since CD147 is expressed in a variety of cells, including epithelial and neuronal cells, this molecule possibly functions as an entry receptor for MeV in SLAM-negative cells. This is the first report among members of the Mononegavirales that CD147 is used as a virus entry receptor via incorporated CypB in the virions.

Benefits of napping and an extended duration of recovery sleep on alertness and immune cells after acute sleep restriction.

PubMed

Faraut, Brice; Boudjeltia, Karim Zouaoui; Dyzma, Michal; Rousseau, Alexandre; David, Elodie; Stenuit, Patricia; Franck, Thierry; Van Antwerpen, Pierre; Vanhaeverbeek, Michel; Kerkhofs, Myriam

2011-01-01

Understanding the interactions between sleep and the immune system may offer insight into why short sleep duration has been linked to negative health outcomes. We, therefore, investigated the effects of napping and extended recovery sleep after sleep restriction on the immune and inflammatory systems and sleepiness. After a baseline night, healthy young men slept for a 2-h night followed by either a standard 8-h recovery night (n=12), a 30-min nap (at 1 p.m.) in addition to an 8-h recovery night (n=10), or a 10-h extended recovery night (n=9). A control group slept 3 consecutive 8-h nights (n=9). Subjects underwent continuous electroencephalogram polysomnography and blood was sampled every day at 7 a.m. Leukocytes, inflammatory and atherogenesis biomarkers (high-sensitivity C-reactive protein, interleukin-8, myeloperoxidase, fibrinogen and apolipoproteins ApoB/ApoA), sleep patterns and sleepiness were investigated. All parameters remained unchanged in the control group. After sleep restriction, leukocyte and - among leukocyte subsets - neutrophil counts were increased, an effect that persisted after the 8-h recovery sleep, but, in subjects who had a nap or a 10-h recovery sleep, these values returned nearly to baseline. Inflammatory and atherogenesis biomarkers were unchanged except for higher myeloperoxidase levels after sleep restriction. The increased sleepiness after sleep restriction was reversed better in the nap and extended sleep recovery conditions. Saliva cortisol decreased immediately after the nap. Our results indicate that additional recovery sleep after sleep restriction provided by a midday nap prior to recovery sleep or a sleep extended night can improve alertness and return leukocyte counts to baseline values. Copyright © 2010 Elsevier Inc. All rights reserved.

Aqueous Phase Synthesis of CuIn Alloy Nanoparticles and Their Application for a CIS (CuInSe2)-Based Printable Solar Battery

PubMed Central

Takahashi, Hideyuki; Fujiki, Hironari; Yokoyama, Shun; Kai, Takayuki; Tohji, Kazuyuki

2018-01-01

To apply CuInSe2 (CIS)-based printable solar batteries; an aqueous phase synthesis method of Cu-In (CI) alloy nanoparticles is studied. Metal complexes in the original solution are restricted to homogenized species by utilizing calculations. For example; [(Cu2+)(ASP2−)2] [ASP: the “body (C4H5O4N)” of aspartic acid (C4H7O4N)] is predominant in the pH 6–13 region (CASP/CCu > 6); while In complexes can be restricted to [(In3+)(OH−)(EDTA4−)] (pH 10–12; CEDTA/CIn = 2) and/or [(In3+)(ASP2−)2] (pH 7–9; CASP/CIn = 5). These results indicate that the added amount of complex reagents should be determined by calculations and not the stoichiometric ratio. The reduction potential of homogenized metal complex is measured by cyclic voltammetry (CV) measurements and evaluated by Nernst’s equation using the overall stability constants. CuIn alloy nanoparticles with a small amount of byproduct (In nanoparticles) are successfully synthesized. The CI precursor films are spin-coated onto the substrate using a 2-propanol dispersion. Then the films are converted into CIS solar cells; which show a maximum conversion efficiency of 2.30%. The relationship between the open circuit potential; short circuit current density; and fill factor indicate that smoothing of the CIS films and improving the crystallinity and thickness increase the solar cell conversion efficiency. PMID:29642413

Effects of hydrogen peroxide on vestibular hair cells in the guinea pig: importance of cell membrane impairment preceding cell death.

PubMed

Tanigawa, Tohru; Tanaka, Hirokazu; Hayashi, Ken; Nakayama, Meiho; Iwasaki, Satoshi; Banno, Shinya; Takumida, Masaya; Brodie, Hirally; Inafuku, Shigeru

2008-11-01

Our findings indicate that oxidative stress induces morphological changes in vestibular hair cells and subsequently leads to cell death after 2.5 h. The aim of this study was to confirm the direct effects of oxidative stress on vestibular hair cells. Vestibular hair cells isolated from guinea pigs were loaded with 1 or 10 mM H2O2, and morphological changes were observed. In addition, in a viability/cytotoxicity assay system, the numbers of dead cells in isolated cristae ampullares were counted 1, 3, and 5 h after loading with H2O2 or artificial perilymph (control). Reactive oxygen, in the form of H2O2, directly affects the cell membrane of isolated vestibular hair cells and causes swelling of the cell body, bleb formation, and shortening of the neck region. Morphological changes occur within 30 min after loading with H2O2, but a significant increase in the number of dead cells is noted only after 3 h.

Silibinin preferentially radiosensitizes prostate cancer by inhibiting DNA repair signaling

PubMed Central

Nambiar, Dhanya K.; Rajamani, Paulraj; Deep, Gagan; Jain, Anil K.; Agarwal, Rajesh; Singh, Rana P.

2015-01-01

Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer (PCa). The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant PCa cell lines by clonogenic, cell cycle, cell death and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μM) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P<0.001) of colony formation selectively in PCa cells, and prolonged and enhanced IR-caused G2/M arrest, apoptosis and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of anti-apoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P<0.01) with higher apoptotic response (10-fold, P<0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced pro-survival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Since silibinin is already in phase II clinical trial for PCa patients, the present finding has translational relevance for radioresistant PCa. PMID:26516160

Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3.

PubMed

Sánchez-Bretaño, Aída; Baba, Kenkichi; Janjua, Uzair; Piano, Ilaria; Gargini, Claudia; Tosini, Gianluca

2017-01-01

Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT 1 and MT 2 ) in 661W cells, then whether MEL can prevent H 2 O 2 -induced cell death, and last, through which pathway MEL confers protection. The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H 2 O 2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H 2 O 2 -mediated cell death, a Fas/FasL antagonist was used before the exposure to H 2 O 2 . Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H 2 O 2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Both MEL receptors (MT 1 and MT 2 ) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H 2 O 2 -mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H 2 O 2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H 2 O 2 , and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H 2 O 2 . The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H 2 O 2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.

Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin.

PubMed

Lu, Pengfei; Chen, Jijun; He, Lixiazi; Ren, Jiangtao; Chen, Haide; Rao, Lingjun; Zhuang, Qinggang; Li, Hui; Li, Lei; Bao, Lei; He, Ji; Zhang, Wei; Zhu, Faming; Cui, Chun; Xiao, Lei

2013-12-01

Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (β2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of β2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.

Antiviral Responses by Swine Primary Bronchoepithelial Cells Are Limited Compared to Human Bronchoepithelial Cells Following Influenza Virus Infection

PubMed Central

Hauser, Mary J.; Dlugolenski, Daniel; Culhane, Marie R.; Wentworth, David E.; Tompkins, S. Mark; Tripp, Ralph A.

2013-01-01

Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment. PMID:23875024

Low abundance of mitochondrial DNA changes mitochondrial status and renders cells resistant to serum starvation and sodium nitroprusside insult.

PubMed

Lee, Sung Ryul; Heo, Hye Jin; Jeong, Seung Hun; Kim, Hyoung Kyu; Song, In Sung; Ko, Kyung Soo; Rhee, Byoung Doo; Kim, Nari; Han, Jin

2015-07-01

Mutation or depletion of mitochondrial DNA (mtDNA) can cause severe mitochondrial malfunction, originating from the mitochondrion itself, or from the crosstalk between nuclei and mitochondria. However, the changes that would occur if the amount of mtDNA is diminished are less known. Thus, we generated rat myoblast H9c2 cells containing lower amounts of mtDNA via ethidium bromide and uridine supplementation. After confirming the depletion of mtDNA by quantitative PCR and gel electrophoresis analysis, we investigated the changes in mitochondrial physical parameters by using flow cytometry. We also evaluated the resistance of these cells to serum starvation and sodium nitroprusside. H9c2 cells with diminished mtDNA contents showed decreased mitochondrial membrane potential, mass, free calcium, and zinc ion contents as compared to naïve H9c2 cells. Furthermore, cytosolic and mitochondrial reactive oxygen species levels were significantly higher in mtDNA-lowered H9c2 cells than in the naïve cells. Although the oxygen consumption rate and cell proliferation were decreased, mtDNA-lowered H9c2 cells were more resistant to serum deprivation and nitroprusside insults than the naïve H9c2 cells. Taken together, we conclude that the low abundance of mtDNA cause changes in cellular status, such as changes in reactive oxygen species, calcium, and zinc ion levels inducing resistance to stress. © 2015 International Federation for Cell Biology.

CD147/EMMPRIN Acts as a Functional Entry Receptor for Measles Virus on Epithelial Cells▿

PubMed Central

Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Terao-Muto, Yuri; Sato, Hiroki; Kai, Chieko

2010-01-01

Measles is a highly contagious human disease caused by measles virus (MeV) and remains the leading cause of death in children, particularly in developing countries. Wild-type MeV preferentially infects lymphocytes by using signaling lymphocytic activation molecule (SLAM), whose expression is restricted to hematopoietic cells, as a receptor. MeV also infects other epithelial and neuronal cells that do not express SLAM and causes pneumonia and diarrhea and, sometimes, serious symptoms such as measles encephalitis and subacute sclerosing panencephalitis. The discrepancy between the tissue tropism of MeV and the distribution of SLAM-positive cells suggests that there are unknown receptors other than SLAM for MeV. Here we identified CD147/EMMPRIN (extracellular matrix metalloproteinase inducer), a transmembrane glycoprotein, which acts as a receptor for MeV on epithelial cells. Furthermore, we found the incorporation of cyclophilin B (CypB), a cellular ligand for CD147, in MeV virions, and showed that inhibition of CypB incorporation significantly attenuated SLAM-independent infection on epithelial cells, while it had no effect on SLAM-dependent infection. To date, MeV infection was considered to be triggered by binding of its hemagglutinin (H) protein and cellular receptors. Our present study, however, indicates that MeV infection also occurs via CD147 and virion-associated CypB, independently of MeV H. Since CD147 is expressed in a variety of cells, including epithelial and neuronal cells, this molecule possibly functions as an entry receptor for MeV in SLAM-negative cells. This is the first report among members of the Mononegavirales that CD147 is used as a virus entry receptor via incorporated CypB in the virions. PMID:20147391

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessedmore » by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS. ► Aquaporin-8 knockdown causes ROS-induced mitochondrial depolarization and cell death. ► Mitochondrial permeability transition blockage prevents depolarization and cell death.« less

Hydrogen Suppresses Hypoxia/Reoxygenation-Induced Cell Death in Hippocampal Neurons Through Reducing Oxidative Stress.

PubMed

Wei, Rong; Zhang, Rufang; Xie, Yewei; Shen, Li; Chen, Fang

2015-01-01

Deep hypothermic circulatory arrest (DHCA) is a cerebral protection technique that has been used in the operations involving the aortic arch and brain aneurysm for decades. We previous showed that DHCA treated rats developed a significant oxidative stress and apoptosis in neurons. We here intend to investigate the protective the effect of hydrogen against oxidative stress-induced cell injury and the involved mechanisms using an in vitro experimental model of hypoxia/reoxygenation (H/R) on HT-22 cells. The model of H/R was established using an airtight culture container and the anaeropack. Measurement of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production was used H2DCFDA and JC-1 staining. Western blot was used for the quantification of Akt, p-Akt, Bcl-2, Bax and cleaved caspase-3 proteins. The microRNA (miRNA) profile in hippocampal neurons from rat model of DHCA was determined by miRNA deep sequencing. The elevation of ROS and reduction of MMP were significantly induced by the treatment with hypoxia for 18 h followed by reoxygenation for 6 h. Hydrogen treatment significantly reduced H/R-caused cell death. The levels of p-Akt (Ser 473) and Bcl-2 were significantly increased while Bax and cleaved caspase-3 were decreased by hydrogen treatment on the model of H/R. The expression of miR-200 family was significantly elevated in model of DHCA and H/R. Hydrogen administration inhibited the H/R-induced expression of miR-200 family in HT-22 cells. In addition, inhibition of miR-200 family suppressed H/R-caused cell death through reducing ROS production. These results suggest that H/R causes oxidative stress-induced cell death and that the hydrogen protects against H/R-induced cell death in HT22 cells, in part, due to reducing expression of miR-200 family. © 2015 S. Karger AG, Basel.

A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model.

PubMed

Zhang, Wei; Zhu, Yao-Hong; Yang, Jin-Cai; Yang, Gui-Yan; Zhou, Dong; Wang, Jiu-Feng

2015-01-01

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-α concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC.

A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model

PubMed Central

Yang, Jin-Cai; Yang, Gui-Yan; Zhou, Dong; Wang, Jiu-Feng

2015-01-01

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-α concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC. PMID:25915861

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.

PubMed

Brass, Abraham L; Huang, I-Chueh; Benita, Yair; John, Sinu P; Krishnan, Manoj N; Feeley, Eric M; Ryan, Bethany J; Weyer, Jessica L; van der Weyden, Louise; Fikrig, Erol; Adams, David J; Xavier, Ramnik J; Farzan, Michael; Elledge, Stephen J

2009-12-24

Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses antiviral restriction factors to defend against infection. To find host cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing. We discovered that the interferon-inducible transmembrane proteins IFITM1, 2, and 3 restrict an early step in influenza A viral replication. The IFITM proteins confer basal resistance to influenza A virus but are also inducible by interferons type I and II and are critical for interferon's virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus and West Nile virus. Collectively this work identifies a family of antiviral restriction factors that mediate cellular innate immunity to at least three major human pathogens. Copyright 2009 Elsevier Inc. All rights reserved.

Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thanan, Raynoo; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002; Techasen, Anchalee

Oxidative stress is a cause of inflammation–related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H{sub 2}O{sub 2}) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H{sub 2}O{sub 2}-resistant cell line from an immortal cholangiocytemore » cell line (MMNK1) by chronic treatment with low-concentration H{sub 2}O{sub 2} (25 μM). After 72 days of induction, H{sub 2}O{sub 2}-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H{sub 2}O{sub 2}-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 μM H{sub 2}O{sub 2}). These findings suggest that H{sub 2}O{sub 2}-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes. - Highlights: • An H{sub 2}O{sub 2}-resistant ox-MMNK1-L cells was established from immortalized cholangiocytes. • The resistance was acquired by daily treatment of low H{sub 2}O{sub 2} (25 μM) for 15 passages. • The cells highly expressed catalase, SODs and DNMT1 with rapid cell proliferation. • Pseudopodia and the loss of cell-to-cell adhesion appeared by 100 μM H{sub 2}O{sub 2} treatment. • The resistant cells can be used as a model of oxidative stress-related carcinogenesis.« less

Phage adsorption to Lactobacillus plantarum: influence of physiological and environmental factors.

PubMed

Marcó, M Briggiler; Reinheimer, J A; Quiberoni, A

2010-04-15

Bacteriophage infection of lactic acid bacteria (LAB) constitutes one of the major problems in the dairy industry, causing economic losses and a constant risk of low quality and/or unsafe foods. The first step in the phage biology is the adsorption on the host cell surface. In a previous study, a remarkable thermal, chemical and photocatalytic resistance was demonstrated by four phages of Lactobacillus plantarum (ATCC 8014-B1, ATCC 8014-B2, FAGK1 and FAGK2). In the present work, these phages were used to characterize the adsorption process on L. plantarum ATCC 8014. Clearly, the characterization of this process could increase the possibilities of design useful strategies in order to prevent phage infections. The influence of Ca(2+), temperature, pH and physiological cell state on phage adsorption was investigated. Burst sizes of phages ATCC 8014-B1 and ATCC 8014-B2 were 60 and 83 PFU/infective centre, respectively. The four phages exhibited a high infectivity even at pH 4 and pH 11. Calcium or magnesium ions were not indispensable for cell lysis and plaque formation, and more than 99% of phage particles were adsorbed either in the presence or absence of Ca(2+), after 15 min at 37 degrees C. Phage adsorption was only partially affected at 50 degrees C, while reached its maximum between 30 and 42 degrees C. The highest adsorption values (99.9%) were observed from pH 5 to 7, after 30 min at 37 degrees C. Adsorption rates decreased after the thermal inactivation of cells, though, when 20 microg/ml of chloramphenicol was used, adsorption values were similar on treated and untreated cells. All these results showed that the adsorption process was only partially affected by a few conditions: thermally killed host cells, an incubation temperature of 50 degrees C and pH values of 9 and 10. Nevertheless, and unfortunately, those conditions are not commonly applied during fermented food manufacturing, thus restricting highly the application of strategies currently available to reduce phage infections in industrial environments. This work also contributes to increase the currently knowledge on the biological aspects of L. plantarum bacteriophages. (c) 2010 Elsevier B.V. All rights reserved.

Restrictive or Liberal Red-Cell Transfusion for Cardiac Surgery.

PubMed

Mazer, C David; Whitlock, Richard P; Fergusson, Dean A; Hall, Judith; Belley-Cote, Emilie; Connolly, Katherine; Khanykin, Boris; Gregory, Alexander J; de Médicis, Étienne; McGuinness, Shay; Royse, Alistair; Carrier, François M; Young, Paul J; Villar, Juan C; Grocott, Hilary P; Seeberger, Manfred D; Fremes, Stephen; Lellouche, François; Syed, Summer; Byrne, Kelly; Bagshaw, Sean M; Hwang, Nian C; Mehta, Chirag; Painter, Thomas W; Royse, Colin; Verma, Subodh; Hare, Gregory M T; Cohen, Ashley; Thorpe, Kevin E; Jüni, Peter; Shehata, Nadine

2017-11-30

The effect of a restrictive versus liberal red-cell transfusion strategy on clinical outcomes in patients undergoing cardiac surgery remains unclear. In this multicenter, open-label, noninferiority trial, we randomly assigned 5243 adults undergoing cardiac surgery who had a European System for Cardiac Operative Risk Evaluation (EuroSCORE) I of 6 or more (on a scale from 0 to 47, with higher scores indicating a higher risk of death after cardiac surgery) to a restrictive red-cell transfusion threshold (transfuse if hemoglobin level was <7.5 g per deciliter, starting from induction of anesthesia) or a liberal red-cell transfusion threshold (transfuse if hemoglobin level was <9.5 g per deciliter in the operating room or intensive care unit [ICU] or was <8.5 g per deciliter in the non-ICU ward). The primary composite outcome was death from any cause, myocardial infarction, stroke, or new-onset renal failure with dialysis by hospital discharge or by day 28, whichever came first. Secondary outcomes included red-cell transfusion and other clinical outcomes. The primary outcome occurred in 11.4% of the patients in the restrictive-threshold group, as compared with 12.5% of those in the liberal-threshold group (absolute risk difference, -1.11 percentage points; 95% confidence interval [CI], -2.93 to 0.72; odds ratio, 0.90; 95% CI, 0.76 to 1.07; P<0.001 for noninferiority). Mortality was 3.0% in the restrictive-threshold group and 3.6% in the liberal-threshold group (odds ratio, 0.85; 95% CI, 0.62 to 1.16). Red-cell transfusion occurred in 52.3% of the patients in the restrictive-threshold group, as compared with 72.6% of those in the liberal-threshold group (odds ratio, 0.41; 95% CI, 0.37 to 0.47). There were no significant between-group differences with regard to the other secondary outcomes. In patients undergoing cardiac surgery who were at moderate-to-high risk for death, a restrictive strategy regarding red-cell transfusion was noninferior to a liberal strategy with respect to the composite outcome of death from any cause, myocardial infarction, stroke, or new-onset renal failure with dialysis, with less blood transfused. (Funded by the Canadian Institutes of Health Research and others; TRICS III ClinicalTrials.gov number, NCT02042898 .).

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

PubMed

Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L; Stein, Paul L; Wang, Chyung-Ru

2014-12-01

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Retino-protective effect of Bucida buceras against oxidative stress induced by H2O2 in human retinal pigment epithelial cells line.

PubMed

Iloki-Assanga, Simon Bernard; Lewis-Luján, Lidianys María; Fernández-Angulo, Daniela; Gil-Salido, Armida Andrea; Lara-Espinoza, Claudia Lizeth; Rubio-Pino, José Luis

2015-07-29

Reactive Oxygen Species (ROS) impair the physiological functions of Retinal Pigment Epithelial (RPE) cells, which are known as one major cause of age-related macular degeneration and retinopathy diseases. The purpose of this study is to explore the cytoprotective effects of the antioxidant Bucida buceras extract in co-treatment with hydrogen peroxide (H2O2) delivery as a single addition or with continuous generation using glucose oxidase (GOx) in ARPE-19 cell cultures. The mechanism of Bucida buceras extract is believed to be associated with their antioxidant capacity to protect cells against oxidative stress. A comparative oxidative stress H2O2-induced was performed by addition and enzymatic generation using glucose oxidase on human retinal pigment epithelial cells line. H2O2-induced injury was measured by toxic effects (cell death and apoptotic pathway) and intracellular redox status: glutathione (GSH), antioxidant enzymes (catalase and glutathione peroxidase) and reducing power (FRAP). The retino-protective effect of co-treatment with Bucida buceras extract on H2O2-induced human RPE cell injury was investigated by cell death (MTT assay) and oxidative stress biomarkers (H2O2, GSH, CAT, GPx and FRAP). Bucida buceras L. extract is believed to be associated with the ability to prevent cellular oxidative stress. When added as a pulse, H2O2 is rapidly depleted and the cytotoxicity analyses show that cells can tolerate short exposure to high peroxide doses delivered as a pulse but are susceptible to lower chronic doses. Co-treatment with Bucida buceras was able to protect the cells against H2O2-induced injury. In addition to preventing cell death treatment with antioxidant plant could also reverse the significant decrease in GSH level, catalase activity and reducing power caused by H2O2. These findings suggest that Bucida buceras could protect RPE against ocular pathogenesis associated with oxidative stress induced by H2O2-delivered by addition and enzymatic generation.

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

PubMed

Doronin, K; Kuppuswamy, M; Toth, K; Tollefson, A E; Krajcsi, P; Krougliak, V; Wold, W S

2001-04-01

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.

Presence and characterization of shiga toxin-producing Escherichia coli and other potentially diarrheagenic E. coli strains in retail meats.

PubMed

Xia, Xiaodong; Meng, Jianghong; McDermott, Patrick F; Ayers, Sherry; Blickenstaff, Karen; Tran, Thu-Thuy; Abbott, Jason; Zheng, Jie; Zhao, Shaohua

2010-03-01

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx(1) and stx(2), 2 positive for stx(1), and 10 positive for stx(2). The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx(2) genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.

Concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in two types of porcine macrophages: apoptosis, production of ROS and formation of multinucleated giant cells.

PubMed

Kavanová, Lenka; Matiašková, Katarína; Levá, Lenka; Štěpánová, Hana; Nedbalcová, Kateřina; Matiašovic, Ján; Faldyna, Martin; Salát, Jiří

2017-05-04

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant and economically important infectious diseases affecting swine worldwide and can predispose pigs to secondary bacterial infections caused by, e.g. Haemophilus parasuis. The aim of the presented study was to compare susceptibility of two different types of macrophages which could be in contact with both pathogens during infection with PRRS virus (PRRSV) and in co-infection with H. parasuis. Alveolar macrophages (PAMs) as resident cells provide one of the first lines of defence against microbes invading lung tissue. On the other hand, monocyte derived macrophages (MDMs) represent inflammatory cells accumulating at the site of inflammation. While PAMs were relatively resistant to cytopathogenic effect caused by PRRSV, MDMs were much more sensitive to PRRSV infection. MDMs infected with PRRSV increased expression of pro-apoptotic Bad, Bax and p53 mRNA. Increased mortality of MDMs may be also related to a higher intensity of ROS production after infection with PRRSV. In addition, MDMs (but not PAMs) infected with H. parasuis alone formed multinucleated giant cells (MGC); these cells were not observed in MDMs infected with both pathogens. Higher sensitivity of MDMs to PRRSV infection, which is associated with limited MDMs survival and restriction of MGC formation, could contribute to the development of multifactorial respiratory disease of swine.

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

PubMed Central

Kane, AB; Stanton, RP; Raymond, EG; Dobson, ME; Knafelc, ME; Farber, JL

1980-01-01

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins. PMID:6161936

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

PubMed Central

Yang, Yang; Durando, Michael; Smith-Roe, Stephanie L.; Sproul, Chris; Greenwalt, Alicia M.; Kaufmann, William; Oh, Sehyun; Hendrickson, Eric A.; Vaziri, Cyrus

2013-01-01

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase. PMID:23295675

Methadone but not morphine inhibits lubiprostone-stimulated Cl- currents in T84 intestinal cells and recombinant human ClC-2, but not CFTR Cl- currents.

PubMed

Cuppoletti, John; Chakrabarti, Jayati; Tewari, Kirti; Malinowska, Danuta H

2013-05-01

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl(-) channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl(-) currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl(-) currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl(-) currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl(-) currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl(-) currents with half-maximal inhibition at 100 and 200-230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl(-) currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl(-) currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl(-) currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl(-) currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.

Identification of a novel HLA-A*02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene

PubMed Central

YOSHIMURA, MAYUKO; TADA, YOSHITAKA; OFUZI, KAZUYA; YAMAMOTO, MASAKAZU; NAKATSURA, TETSUYA

2014-01-01

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A*02:01- and HLA-A*24:02-restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK-derived candidate peptides for the induction of tumor-reactive CTLs. Nine EML4-ALK-derived peptides were selected by a computer algorithm based on a permissive HLA-A*02:01 or HLA-A*24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide-specific CTL clone. This CTL clone specifically recognized peptide-pulsed T2 cells and H2228 cells expressing HLA-A*02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA-class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A*02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4-ALK-positive cancers. PMID:24842630

Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene.

PubMed

Yoshimura, Mayuko; Tada, Yoshitaka; Ofuzi, Kazuya; Yamamoto, Masakazu; Nakatsura, Tetsuya

2014-07-01

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A 02:01- and HLA-A 24:02‑restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK‑derived candidate peptides for the induction of tumor‑reactive CTLs. Nine EML4-ALK‑derived peptides were selected by a computer algorithm based on a permissive HLA-A 02:01 or HLA-A 24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide‑specific CTL clone. This CTL clone specifically recognized peptide‑pulsed T2 cells and H2228 cells expressing HLA-A 02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA‑class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A 02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4‑ALK-positive cancers.

Food restriction enhances oxidative status in aging rats with neuroprotective effects on myenteric neuron populations in the proximal colon.

PubMed

Schoffen, João Paulo Ferreira; Santi Rampazzo, Ana Paula; Cirilo, Carla Possani; Zapater, Mariana Cristina Umada; Vicentini, Fernando Augusto; Comar, Jurandir Fernando; Bracht, Adelar; Natali, Maria Raquel Marçal

2014-03-01

Food restriction may slow the aging process by increasing the levels of antioxidant defenses and reducing cell death. We evaluated the effects of food restriction on oxidative and nutritional status, myenteric cell populations, and the colonic muscle layer in aging rats. Wistar rats were distributed into control groups (7, 12, and 23months of age) and subjected to food restriction (50% of normal diet) beginning at 7months of age. The animals were sacrificed, and blood was collected to evaluate its components and markers of oxidative status, including thiobarbituric acid-reactive substances, reduced glutathione, catalase, glutathione peroxidase, and total antioxidant capacity. The proximal colon was collected to evaluate HuC/D and neuronal nitric oxide synthase (nNOS)-positive and -negative myenteric neurons, S-100 glial cells, and the muscle layer. Age negatively affected oxidative status in the animals, which also increased the levels of total cholesterol, protein, and globulins and increased the thickness of the muscle layer. Aging also reduced the number and hypertrophied glial cell bodies, HuC/D neurons, and nNOS-negative and -positive neurons. An improvement was observed in oxidative status and the levels of total cholesterol and triglycerides with food restriction, which also provided neuroprotection of the intrinsic innervation. However, food restriction accentuated the loss of enteric glia and caused hypertrophy in the muscle layer at 23months. Food restriction improved oxidative and nutritional status in rats and protected HuC/D neurons and nNOS-negative and -positive neurons against neuronal loss. Nevertheless, food restriction caused morphoquantitative changes in glial cell populations, with possible interference with colonic neuromuscular control. Copyright © 2014 Elsevier Inc. All rights reserved.

Mechanisms of hypochlorite injury of target cells.

PubMed Central

Schraufstätter, I U; Browne, K; Harris, A; Hyslop, P A; Jackson, J H; Quehenberger, O; Cochrane, C G

1990-01-01

HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants. PMID:2153710

Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells.

PubMed

Vacharaksa, Anjalee; Asrani, Anil C; Gebhard, Kristin H; Fasching, Claudine E; Giacaman, Rodrigo A; Janoff, Edward N; Ross, Karen F; Herzberg, Mark C

2008-07-17

Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells) were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Deltaenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs) or MOLT4 cells (CD4+ CCR5+) by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

A Membrane-Free Neutral pH Formate Fuel Cell Enabled by a Selective Nickel Sulfide Oxygen Reduction Catalyst.

PubMed

Yan, Bing; Concannon, Nolan M; Milshtein, Jarrod D; Brushett, Fikile R; Surendranath, Yogesh

2017-06-19

Polymer electrolyte membranes employed in contemporary fuel cells severely limit device design and restrict catalyst choice, but are essential for preventing short-circuiting reactions at unselective anode and cathode catalysts. Herein, we report that nickel sulfide Ni 3 S 2 is a highly selective catalyst for the oxygen reduction reaction in the presence of 1.0 m formate. We combine this selective cathode with a carbon-supported palladium (Pd/C) anode to establish a membrane-free, room-temperature formate fuel cell that operates under benign neutral pH conditions. Proof-of-concept cells display open circuit voltages of approximately 0.7 V and peak power values greater than 1 mW cm -2 , significantly outperforming the identical device employing an unselective platinum (Pt) cathode. The work establishes the power of selective catalysis to enable versatile membrane-free fuel cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation, characterization and preclinical development of human glial-restricted progenitor cells for treatment of neurological disorders.

PubMed

Sandrock, Robert W; Wheatley, Will; Levinthal, Cynthia; Lawson, Jennifer; Hashimoto, Brooke; Rao, Mahendra; Campanelli, James T

2010-05-01

Glial-restricted progenitor cells (GRPs), a neural cell population that gives rise to astrocytes and oligodendrocytes both in vitro and in vivo, hold great promise as a cellular therapeutic for the treatment of demyelinating and neurodegenerative diseases of the CNS. The manufacturing and characterization protocols of human-derived GRPs (hGRPs; trade name Q-Cells) for use in a clinical setting that adhere to rigorous standards for their isolation, propagation, characterization and storage are presented. hGRPs, defined by their immunoreactivity with A2B5 antibodies, were isolated from fetal cadaver forebrain tissue of mice 17-24 weeks gestational age using Miltenyi paramagnetic bead cell separation technology. GRPs were grown in a defined xenobiotic-free medium for 6 days. At harvest, hGRPs were characterized using immunocytochemical techniques. Long-term cryopreservation and storage conditions, and viability upon freeze-thaw were determined. The phenotypic differentiation potential of hGRPs was determined by implantation experiments into the CNS of shiverer mice. hGRPs were isolated from over 50 neural tissues of either sex during gestational ages of 17-24 weeks. Cells expanded out to 6 days in vitro in a xenobiotic-free medium demonstrated very consistent immunocytochemical profiles. No residual antibody used in the purification process was detected after 6 days of growth in vitro. GRPs could be frozen at up to 24 million cells/ml and were over 70% viable upon freeze-thaw. Thawed hGRPs transplanted into the brain of the dysmyelinated shiverer mouse model were observed to differentiate into both glial fibrillary acidic protein-positive astrocytes and myelin basic protein-positive oligodendrocytes; no human-derived NeuN-positive neuronal cells were observed and no abnormal cell proliferation was observed. We demonstrate that hGRPs can be consistently obtained, propagated, cryopreserved and characterized using protocols that can be transferred to a good laboratory practice/good manufacturing practice setting for the manufacture of clinical-grade hGRP cellular therapeutics. Functional data demonstrate that cells manufactured under these conditions are able to differentiate into appropriate cellular phenotypes in an animal model of dysmyelination.

Epigallocatechin gallate (EGCG) prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells.

PubMed

Cia, David; Vergnaud-Gauduchon, Juliette; Jacquemot, Nathalie; Doly, Michel

2014-09-01

To determine whether the green tea polyphenol epigallocatechin gallate (EGCG) could prevent H(2)O(2)-induced oxidative stress in primary rat retinal pigment epithelial cells. Primary cultures of retinal pigment epithelium (RPE) cells were established from Long-Evans newborn rats. RPE cells were pretreated with various concentrations of EGCG for 24 h before being exposed to hydrogen peroxide (H(2)O(2)) for 2 h to induce oxidative stress. Cell metabolic activity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was quantified by flow cytometry using propidium iodide (PI). Treatment of RPE cells with EGCG alone does not affect the cell viability up to 50 µM. Exposure of RPE cells to 600 µM H(2)O(2) caused a significant decrease in cell viability; whereas pretreatment with 10, 25, and 50 µM EGCG significantly reduced this decrease in a dose-dependent manner. The proportion of PI-positive cells increased significantly in cultures treated with H(2)O(2) alone; whereas pretreatment of RPE cells with 50 µM EGCG significantly reduced H(2)O(2)-induced RPE cell death. Our study shows that EGCG pretreatment can protect primary rat RPE cells from H(2)O(2)-induced death. This suggests potential effect of EGCG in the prevention of retinal diseases associated with H(2)O(2)-induced oxidative stress.

Nonlinear feedback drives homeostatic plasticity in H2O2 stress response

PubMed Central

Goulev, Youlian; Morlot, Sandrine; Matifas, Audrey; Huang, Bo; Molin, Mikael; Toledano, Michel B; Charvin, Gilles

2017-01-01

Homeostatic systems that rely on genetic regulatory networks are intrinsically limited by the transcriptional response time, which may restrict a cell’s ability to adapt to unanticipated environmental challenges. To bypass this limitation, cells have evolved mechanisms whereby exposure to mild stress increases their resistance to subsequent threats. However, the mechanisms responsible for such adaptive homeostasis remain largely unknown. Here, we used live-cell imaging and microfluidics to investigate the adaptive response of budding yeast to temporally controlled H2O2 stress patterns. We demonstrate that acquisition of tolerance is a systems-level property resulting from nonlinearity of H2O2 scavenging by peroxiredoxins and our study reveals that this regulatory scheme induces a striking hormetic effect of extracellular H2O2 stress on replicative longevity. Our study thus provides a novel quantitative framework bridging the molecular architecture of a cellular homeostatic system to the emergence of nonintuitive adaptive properties. DOI: http://dx.doi.org/10.7554/eLife.23971.001 PMID:28418333

Phenanthrene exposure induces cardiac hypertrophy via reducing miR-133a expression by DNA methylation

PubMed Central

Huang, Lixing; Xi, Zhihui; Wang, Chonggang; Zhang, Youyu; Yang, Zhibing; Zhang, Shiqi; Chen, Yixin; Zuo, Zhenghong

2016-01-01

Growing evidence indicates that there is an emerging link between environmental pollution and cardiac hypertrophy, while the mechanism is unclear. The objective of this study was to examine whether phenanthrene (Phe) could cause cardiac hypertrophy, and elucidate the molecular mechanisms involved. We found that: 1) Phe exposure increased the heart weight and cardiomyocyte size of rats; 2) Phe exposure led to enlarged cell size, and increased protein synthesis in H9C2 cells; 3) Phe exposure induced important markers of cardiac hypertrophy, such as atrial natriuretic peptide, B-type natriuretic peptide, and c-Myc in H9C2 cells and rat hearts; 4) Phe exposure perturbed miR-133a, CdC42 and RhoA, which were key regulators of cardiac hypertrophy, in H9C2 cells and rat hearts; 5) Phe exposure induced DNA methyltransferases (DNMTs) in H9C2 cells and rat hearts; 6) Phe exposure led to methylation of CpG sites within the miR-133a locus and reduced miR-133a expression in H9C2 cells; 7) DNMT inhibition and miR-133a overexpression could both alleviate the enlargement of cell size and perturbation of CdC42 and RhoA caused by Phe exposure. These results indicated that Phe could induce cardiomyocyte hypertrophy in the rat and H9C2 cells. The mechanism might involve reducing miR-133a expression by DNA methylation. PMID:26830171

AFRRI Reports, Fourth Quarter 1991

DTIC Science & Technology

1992-01-01

Gallin, E. K. Heat induces intracellular acidification in human A-431 cells : role of Na’-H’ exchange and metabolism. SR91-49: MacVittie, T. J., Monroy, R... induced release of DNA front agarose plugs treated with Not I restriction enzyme As above, wild-type and strain 112 cells were exposed to 2 kGy and...times. Ager and co-workers (1990) found that following irradiation of CHO-KI cells , replication forks showed reduced PFGE- induced migration out of the

Influence of environmental pH on G2-phase arrest caused by ionizing radiation.

PubMed

Park, Heon Joo; Lee, Sang Hwa; Chung, HyunSook; Rhee, Yun Hee; Lim, Byung Uk; Ha, Sung Whan; Griffin, Robert J; Lee, Hyung Sik; Song, Chang Won; Choi, Eun Kyung

2003-01-01

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

Evaluation of imazethapyr-induced DNA oxidative damage by alkaline Endo III- and Fpg-modified single-cell gel electrophoresis assay in Hypsiboas pulchellus tadpoles (Anura, Hylidae).

PubMed

Pérez-Iglesias, Juan Manuel; Ruiz de Arcaute, Celeste; Natale, Guillermo S; Soloneski, S; Larramendy, Marcelo L

2017-08-01

Imazethapyr (IMZT) is a selective postemergent herbicide with residual action. Available data analyzing its effects in aquatic vertebrates are scarce. In previous studies, we demonstrated that IMZT induces lesions into the DNA of Hypsiboas pulchellus tadpoles using the single-cell gel electrophoresis (SCGE) assay as a biomarker for genotoxicity. Currently, this assay can be modified by including incubation with lesion-specific endonucleases, e.g., endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which detect oxidized pyrimidine and purine bases, respectively. The aim of this study was to evaluate the role of oxidative stress in the genotoxic damage in circulating blood cells of H. pulchellus tadpoles exposed to the IMZT-based Pivot H ® formulation (10.59% IMZT) at a concentration equivalent to 25% of the LC 50 (96h) value (0.39mg/L IMZT) during 48 and 96h. Our results demonstrate that the herbicide induces oxidative DNA damage on H. pulchellus tadpoles at purines bases but not at pyrimidines. Our findings represent the first evidence of oxidative damage caused by IMZT on anuran DNA using the alkaline restriction enzyme-modified SCGE assay. Copyright © 2017 Elsevier Inc. All rights reserved.

pH-Dependent Antimicrobial Properties of Copper Oxide Nanoparticles in Staphylococcus aureus

PubMed Central

Hsueh, Yi-Huang; Tsai, Ping-Han; Lin, Kuen-Song

2017-01-01

The antimicrobial properties of CuO nanoparticles have been investigated, but the underlying mechanisms of toxicity remain the subject of debate. Here, we show that CuO nanoparticles exhibit significant toxicity at pH 5 against four different Staphylococcus aureus (S. aureus) strains, including Newman, SA113, USA300, and ATCC6538. At this pH, but not at pH 6 and 7, 5 mM CuO nanoparticles effectively caused reduction of SA113 and Newman cells and caused at least 2 log reduction, whereas 20 mM killed most strains but not USA300. At 5 mM, the nanoparticles were also found to dramatically decrease reductase activity in SA113, Newman, and ATCC6538 cells, but not USA300 cells. In addition, analysis of X-ray absorption near-edge structure and extended X-ray absorption fine structure confirmed that S. aureus cells exposed to CuO nanoparticles contain CuO, indicating that Cu2+ ions released from nanoparticles penetrate bacterial cells and are subsequently oxidized intracellularly to CuO at mildly acidic pH. The CuO nanoparticles were more soluble at pH 5 than at pH 6 and 7. Taken together, the data conclusively show that the toxicity of CuO nanoparticles in mildly acidic pH is caused by Cu2+ release, and that USA300 is more resistant to CuO nanoparticles (NPs) than the other three strains. PMID:28397766

Molecular mechanisms behind the accumulation of adenosine triphosphate (ATP) and H2O2 in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection

USDA-ARS?s Scientific Manuscript database

Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells,...

Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

PubMed Central

Barbaric, Ivana; Biga, Veronica; Gokhale, Paul J.; Jones, Mark; Stavish, Dylan; Glen, Adam; Coca, Daniel; Andrews, Peter W.

2014-01-01

Summary Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns. PMID:25068128

CD4+ T Cells Reactive to Enteric Bacterial Antigens in Spontaneously Colitic C3H/HeJBir Mice: Increased T Helper Cell Type 1 Response and Ability to Transfer Disease

PubMed Central

Cong, Yingzi; Brandwein, Steven L.; McCabe, Robert P.; Lazenby, A.; Birkenmeier, Edward H.; Sundberg, John P.; Elson, Charles O.

1998-01-01

C3H/HeJBir mice are a new substrain that spontaneously develop colitis early in life. This study was done to determine the T cell reactivity of C3H/HeJBir mice to candidate antigens that might be involved in their disease. C3H/HeJBir CD4+ T cells were strongly reactive to antigens of the enteric bacterial flora, but not to epithelial or food antigens. The stimulatory material in the enteric bacteria was trypsin sensitive and restricted by class II major histocompatibility complex molecules, but did not have the properties of a superantigen. The precursor frequency of interleuken (IL)-2–producing, bacterial-reactive CD4+ T cells in colitic mice was 1 out of 2,000 compared to 1 out of 20,000–25,000 in noncolitic control mice. These T cells produced predominately IL-2 and interferon γ, consistent with a T helper type 1 cell response and were present at 3–4 wk, the age of onset of the colitis. Adoptive transfer of bacterial-antigen–activated CD4+ T cells from colitic C3H/HeJBir but not from control C3H/HeJ mice into C3H/HeSnJ scid/scid recipients induced colitis. These data represent a direct demonstration that T cells reactive with conventional antigens of the enteric bacterial flora can mediate chronic inflammatory bowel disease. PMID:9500788

A hypoxia- and {alpha}-fetoprotein-dependent oncolytic adenovirus exhibits specific killing of hepatocellular carcinomas.

PubMed

Kwon, Oh-Joon; Kim, Pyung-Hwan; Huyn, Steven; Wu, Lily; Kim, Minjung; Yun, Chae-Ok

2010-12-15

Oncolytic adenoviruses (Ad) constitute a new promising modality of cancer gene therapy that displays improved efficacy over nonreplicating Ads. We have previously shown that an E1B 19-kDa-deleted oncolytic Ad exhibits a strong cell-killing effect but lacks tumor selectivity. To achieve hepatoma-restricted cytotoxicity and enhance replication of Ad within the context of tumor microenvironment, we used a modified human α-fetoprotein (hAFP) promoter to control the replication of Ad with a hypoxia response element (HRE). We constructed Ad-HRE(6)/hAFPΔ19 and Ad-HRE(12)/hAFPΔ19 that incorporated either 6 or 12 copies of HRE upstream of promoter. The promoter activity and specificity to hepatoma were examined by luciferase assay and fluorescence-activated cell sorting analysis. In addition, the AFP expression- and hypoxia-dependent in vitro cytotoxicity of Ad-HRE(6)/hAFPΔ19 and Ad-HRE(12)/hAFPΔ19 was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cytopathic effect assay. In vivo tumoricidal activity on subcutaneous and liver orthotopic model was monitored by noninvasive molecular imaging. Ad-HRE(12)/hAFPΔ19 exhibited enhanced tumor selectivity and cell-killing activity when compared with Ad-hAFPΔ19. The tumoricidal activity of Ad-HRE(12)/hAFPΔ19 resulted in significant inhibition of tumor growth in both subcutaneous and orthotopic models. Histologic examination of the primary tumor after treatment confirmed accumulation of viral particles near hypoxic areas. Furthermore, Ad-HRE(12)/hAFPΔ19 did not cause severe inflammatory immune response and toxicity after systemic injection. The results presented here show the advantages of incorporating HREs into a hAFP promoter-driven oncolytic virus. This system is unique in that it acts in both a tissue-specific and tumor environment-selective manner. The greatly enhanced selectivity and tumoricidal activity of Ad-HRE(12)/hAFPΔ19 make it a promising therapeutic agent in the treatment of liver cancers. ©2010 AACR.

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

PubMed

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.

Enhancement of human neural stem cell self-renewal in 3D hypoxic culture.

PubMed

Ghourichaee, Sasan Sharee; Powell, Elizabeth M; Leach, Jennie B

2017-05-01

The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self-renewing and multipotent characteristics, possess enormous tissue-specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well-defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O 2 ) versus hypoxic (3% O 2 ) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096-1106. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Molecular hydrogen protects against oxidative stress-induced SH-SY5Y neuroblastoma cell death through the process of mitohormesis.

PubMed

Murakami, Yayoi; Ito, Masafumi; Ohsawa, Ikuroh

2017-01-01

Inhalation of molecular hydrogen (H2) gas ameliorates oxidative stress-induced acute injuries in the brain. Consumption of water nearly saturated with H2 also prevents chronic neurodegenerative diseases including Parkinson's disease in animal and clinical studies. However, the molecular mechanisms underlying the remarkable effect of a small amount of H2 remain unclear. Here, we investigated the effect of H2 on mitochondria in cultured human neuroblastoma SH-SY5Y cells. H2 increased the mitochondrial membrane potential and the cellular ATP level, which were accompanied by a decrease in the reduced glutathione level and an increase in the superoxide level. Pretreatment with H2 suppressed H2O2-induced cell death, whereas post-treatment did not. Increases in the expression of anti-oxidative enzymes underlying the Nrf2 pathway in H2-treated cells indicated that mild stress caused by H2 induced increased resistance to exacerbated oxidative stress. We propose that H2 functions both as a radical scavenger and a mitohormetic effector against oxidative stress in cells.

Effects of nicotine on cellular proliferation, macromolecular synthesis and cell cycle phase distribution in human and murine cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konno, S.; Chiao, J.; Rossi, J.

1986-05-01

Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in established human and murine cells. In the human promyelocytic HL-60 leukemic cells, 3 mM nicotine results in a 50% inhibition of cellular proliferation after 80 h. Nicotine was also found to affect the cell cycle distribution of HL-60 cells. Treatment with 4 mM nicotine for 20 h causes an increase in proportion of Gl-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes cell arrest in the Gl-phase which may inmore » part account for its effects on cell growth. To determine whether nicotine has a primary effect on the uptake/transport of macromolecular precursors into cells, HL-60 cells were treated with 2-6 mM nicotine for 30 h/sub 3/ at the end of which time cells were labeled with (/sup 3/H)thymidine, (/sup 3/H)uridine, (/sup 14/C)lysine and (/sup 35/S)methionine, the trichloroacetic acid (TCA) soluble and insoluble radioactivities from each of the labeling conditions were determined. These studies show that nicotine primarily affect the synthesis of proteins.« less

Ammonium is a key determinant on the dietary restriction of yeast chronological aging in culture medium

PubMed Central

Santos, Júlia; Leitão-Correia, Fernanda

2015-01-01

New evidences have recently emerged from studies in yeast and in higher eukaryotes showing the importance of nutrient balance in dietary regimes and its effects on longevity regulation. We have previously shown that manipulation ofammoniumconcentration in the culture and/or aging medium can drastically affect chronological lifespan (CLS) of Saccharomyces cerevisiae, especially in amino acid restricted cells. Here we describe that the CLS shortening under amino acid restriction can be completely reverted by removing ammonium from the culture medium. Furthermore, the absence of ammonium, and of any rich nitrogen source, was so effective in extending CLS that no beneficial effect could be observed by further imposing calorie restriction conditions. When present in the culture medium,ammoniumimpaired the consumption of theauxotrophy-complementing amino acidsand caused in an improper cell cycle arrest of the culture. TOR1 deletion reverted ammonium effects both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to have only a milder effect in the mediation ofammonium toxicity under amino acid restriction and no effect on non-restricted cultures. Our studies highlight ammonium as a key effector in the nutritional equilibrium between rich and essential nitrogen sources and glucose required for longevity promotion. PMID:25576917

Cell-type–restricted anti-cytokine therapy: TNF inhibition from one pathogenic source

PubMed Central

Efimov, Grigory A.; Kruglov, Andrei A.; Khlopchatnikova, Zoya V.; Rozov, Fedor N.; Mokhonov, Vladislav V.; Rose-John, Stefan; Scheller, Jürgen; Gordon, Siamon; Stacey, Martin; Drutskaya, Marina S.; Tillib, Sergei V.; Nedospasov, Sergei A.

2016-01-01

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type–restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)—a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine–induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF. PMID:26936954

MHC class I D(k) expression in hematopoietic and nonhematopoietic cells confers natural killer cell resistance to murine cytomegalovirus.

PubMed

Xie, Xuefang; Stadnisky, Michael D; Coats, Ebony R; Ahmed Rahim, Mir Munir; Lundgren, Alyssa; Xu, Wenhao; Makrigiannis, Andrew P; Brown, Michael G

2010-05-11

NK cell-mediated murine cytomegalovirus (MCMV) resistance (Cmv(r)) is under H-2(k) control in MA/My mice, but the underlying gene(s) is unclear. Prior genetic analysis mapped Cmv(r) to the MHC class I (MHC-I) D(k) gene interval. Because NK cell receptors are licensed by and responsive to MHC class I molecules, D(k) itself is a candidate gene. A 10-kb genomic D(k) fragment was subcloned and microinjected into MCMV-susceptible (Cmv(s)) (MA/My.L-H2(b) x C57L)F(1) or (B6 x DBA/2)F(2) embryos. Transgenic founders, which are competent for D(k) expression and germline transgene transmission, were identified and further backcrossed to MA/My.L-H2(b) or C57L mice. Remarkably, D(k) expression delivered NK-mediated resistance in either genetic background. Further, NK cells with cognate inhibitory Ly49G receptors for self-MHC-I D(k) were licensed and critical in protection against MCMV infection. In radiation bone marrow chimeras, NK resistance was significantly diminished when MHC-I D(k) expression was restricted to only hematopoietic or nonhematopoietic cells. Thus, MHC-I D(k) is the H-2(k)-linked Cmv(r) locus; these findings suggest a role for NK cell interaction with D(k)-bearing hematopoietic and nonhematopoietic cells to shape NK-mediated virus immunity.

Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin.

PubMed

Barton, Lacy J; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

2016-07-01

The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. Here we present our studies of one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). We demonstrate that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. We show germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, we investigate the requirement of Ote in the male fertility. We show that ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, our studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, our data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro responses to avian influenza H5 by human CD4 T-cells*

PubMed Central

Cusick, Matthew F; Wang, Shuping; Eckels, David D

2009-01-01

To address the question of whether human T-cells are capable of recognizing novel isolates of influenza virus, in vitro responses to recombinant antigens and synthetic peptides derived from the sequences of H1, H3, and H5 were examined in a cohort of 64 individuals selected from a healthy blood donor population. Humans respond in vitro to H1 and H3 following exposure through natural infection and vaccination. Responses to H5 were well correlated with those to H1 or H3 and thus a significant repertoire of H5-responsive T-cells is present in many individuals; clear non-responders to H1, H3, and H5, however, do exist. Differences were observed in the cytokine responses to H1, H3, and H5; whereas both IL-2 and IFN-γ production characteristic of memory responses were observed for H1 and H3, H5-specific responses elicited primarily IL-2 and little or no IFN-γ consistent with a naïve T cell phenotype. Responses to all influenza HA were restricted by HLA-DR molecules. To address the structural basis for T-cell recognition of H1 and H5, overlapping synthetic peptides were used to identify epitopes and to determine whether recognition of H5 was limited to homologous sequences in H1, the most closely related HA phylogenetically. Although responses were generally correlated, no complete structural overlap was observed. These results suggest that helper T cell cross reactivity between different influenza strains may impart cross-protection to H5N1 strain of influenza. PMID:19841175

NO and H2O2 contribute to SO2 toxicity via Ca2+ signaling in Vicia faba guard cells.

PubMed

Yi, Min; Bai, Heli; Xue, Meizhao; Yi, Huilan

2017-04-01

NO and H 2 O 2 have been implicated as important signals in biotic and abiotic stress responses of plants to the environment. Previously, we have shown that SO 2 exposure increased the levels of NO and H 2 O 2 in plant cells. We hypothesize that, as signaling molecules, NO and H 2 O 2 mediate SO 2 -caused toxicity. In this paper, we show that SO 2 hydrates caused guard cell death in a concentration-dependent manner in the concentration range of 0.25 to 6 mmol L -1 , which was associated with elevation of intracellular NO, H 2 O 2 , and Ca 2+ levels in Vicia faba guard cells. NO donor SNP enhanced SO 2 toxicity, while NO scavenger c-PTIO and NO synthesis inhibitors L-NAME and tungstate significantly prevented SO 2 toxicity. ROS scavenger ascorbic acid (AsA) and catalase (CAT), Ca 2+ chelating agent EGTA, and Ca 2+ channel inhibitor LaCl 3 also markedly blocked SO 2 toxicity. In addition, both c-PTIO and AsA could completely block SO 2 -induced elevation of intracellular Ca 2+ level. Moreover, c-PTIO efficiently blocked SO 2 -induced H 2 O 2 elevation, and AsA significantly blocked SO 2 -induced NO elevation. These results indicate that extra NO and H 2 O 2 are produced and accumulated in SO 2 -treated guard cells, which further activate Ca 2+ signaling to mediate SO 2 toxicity. Our findings suggest that both NO and H 2 O 2 contribute to SO 2 toxicity via Ca 2+ signaling.

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

PubMed Central

Lima, Rogério Barbosa; Salvador, Victor Hugo; dos Santos, Wanderley Dantas; Bubna, Gisele Adriana; Finger-Teixeira, Aline; Soares, Anderson Ricardo; Marchiosi, Rogério; Ferrarese, Maria de Lourdes Lucio; Ferrarese-Filho, Osvaldo

2013-01-01

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth. PMID:24312480

Silencing of the Wnt transcription factor TCF4 sensitizes colorectal cancer cells to (chemo-) radiotherapy

PubMed Central

Kendziorra, Emil; Ahlborn, Kerstin; Spitzner, Melanie; Rave-Fränk, Margret; Emons, Georg; Gaedcke, Jochen; Kramer, Frank; Wolff, Hendrik A.; Becker, Heinz; Beissbarth, Tim; Ebner, Reinhard; Ghadimi, B.Michael; Pukrop, Tobias; Ried, Thomas; Grade, Marian

2011-01-01

A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G2/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy. PMID:21983179

Memory B lymphocytes determine repertoire oligoclonality early after haematopoietic stem cell transplantation

PubMed Central

OMAZIC, B; LUNDKVIST, I; MATTSSON, J; PERMERT, J; NÄSMAN-BJÖRK, I

2003-01-01

The objective of this study was to investigate if oligoclonality of the Ig repertoire post-haematopoietic stem cell transplantation (HSCT) is restricted to memory B lymphocytes or if it is a general property among B lymphocytes. As a measure of B lymphocyte repertoire diversity, we have analysed size distribution of polymerase chain reaction (PCR) amplified Ig H complementarity determining region 3 (CDR3) in naive and memory B lymphocytes isolated from patients before HSCT and at 3, 6 and 12 months after HSCT as well as from healthy controls. We demonstrate a limited variation of the IgH CDR3 repertoire in the memory B lymphocyte population compared to the naive B cell population. This difference was significant at 3 and 6 months post-HSCT. Compared to healthy controls there is a significant restriction of the memory B lymphocyte repertoire at 3 months after HSCT, but not of the naive B lymphocyte repertoire. Twelve months after HSCT, the IgH CDR3 repertoire in both memory and naive B lymphocytes are as diverse as in healthy controls. Thus, our findings suggest a role for memory B cells in the restriction of the oligoclonal B cell repertoire observed early after HSCT, which may be of importance when considering reimmunization of transplanted patients. PMID:12974769

Higher titers of some H5N1 and recent human H1N1 and H3N2 influenza viruses in Mv1 Lu vs. MDCK cells

PubMed Central

2011-01-01

Background The infectivity of influenza A viruses can differ among the various primary cells and continuous cell lines used for such measurements. Over many years, we observed that all things equal, the cytopathic effects caused by influenza A subtype H1N1, H3N2, and H5N1 viruses were often detected earlier in a mink lung epithelial cell line (Mv1 Lu) than in MDCK cells. We asked whether virus yields as measured by the 50% tissue culture infectious dose and plaque forming titer also differed in MDCK and Mv1 Lu cells infected by the same influenza virus subtypes. Results The 50% tissue culture infectious dose and plaque forming titer of many influenza A subtype H1N1, H3N2, and H5N1 viruses was higher in Mv1 Lu than in MDCK cells. Conclusions The yields of influenza subtype H1N1, H3N2, and H5N1 viruses can be higher in Mv1 Lu cells than in MDCK cells. PMID:21314955

Reduced Cystathionine γ-Lyase and Increased miR-21 Expression Are Associated with Increased Vascular Resistance in Growth-Restricted Pregnancies

PubMed Central

Cindrova-Davies, Tereza; Herrera, Emilio A.; Niu, Youguo; Kingdom, John; Giussani, Dino A.; Burton, Graham J.

2013-01-01

Increased vascular impedance in the fetoplacental circulation is associated with fetal hypoxia and growth restriction. We sought to investigate the role of hydrogen sulfide (H2S) in regulating vasomotor tone in the fetoplacental vasculature. H2S is produced endogenously by catalytic activity of cystathionine β-synthase and cystathionine γ-lyase (CSE). Immunohistochemical analysis localized CSE to smooth muscle cells encircling arteries in stem villi. Immunoreactivity was reduced in placentas from pregnancies with severe early-onset growth-restriction and preeclampsia displaying abnormal umbilical artery Doppler waveforms compared with preeclamptic placentas with normal waveforms and controls. These findings were confirmed at the protein and mRNA levels. MicroRNA-21, which negatively regulates CSE expression, was increased in placentas with abnormal Doppler waveforms. Exposure of villus explants to hypoxia-reoxygenation significantly reduced CSE protein and mRNA and increased microRNA-21 expression. No changes were observed in cystathionine β-synthase expression, immunolocalized principally to the trophoblast, in pathologic placentas or in vitro. Finally, perfusion of normal placentas with an H2S donor, after preconstriction with a thromboxane mimetic, resulted in dose-dependent vasorelaxation. Glibenclamide and NG-nitro-l-arginine methyl ester partially blocked the effect, indicating that H2S acts through ATP-sensitive K+ channels and nitric oxide synthesis. These results demonstrate that H2S is a powerful vasodilator of the placental vasculature and that expression of CSE is reduced in placentas associated with increased vascular resistance. PMID:23410520

First Molecular Characterization of Hypoderma actaeon in Cattle and Red Deer (Cervus elaphus) in Portugal.

PubMed

Ahmed, Haroon; Sousa, Sérgio Ramalho; Simsek, Sami; Anastácio, Sofia; Kilinc, Seyma Gunyakti

2017-12-01

Hypoderma spp. larvae cause subcutaneous myiasis in several animal species. The objective of the present investigation was to identify and characterize morphologically and molecularly the larvae of Hypoderma spp. collected from cattle (Bos taurus taurus) and red deer (Cervus elaphus) in the district of Castelo Branco, Portugal. For this purpose, a total of 8 larvae were collected from cattle (n=2) and red deer (n=6). After morphological identification of Hypoderma spp. larvae, molecular characterization was based on PCR-RFLP and mitochondrial CO1 gene sequence analysis. All larvae were morphologically characterized as the third instar larvae (L3) of H. actaeon. Two restriction enzymes were used for molecular identification of the larvae. TaqI restriction enzyme was not able to cut H. actaeon. However, MboII restriction enzyme differentiated Hypoderma species showing 210 and 450 bp bands in H. actaeon. Furthermore, according to the alignment of the mt-CO1 gene sequences of Hypoderma species and to PCR-RFLP findings, all the identified Hypoderma larvae were confirmed as H. actaeon. This is the first report of identification of Hypoderma spp. (Diptera; Oestridae) from cattle and red deer in Portugal, based on morphological and molecular analyses.

Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling.

PubMed

Nambiar, Dhanya K; Rajamani, Paulraj; Deep, Gagan; Jain, Anil K; Agarwal, Rajesh; Singh, Rana P

2015-12-01

Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μmol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer. ©2015 American Association for Cancer Research.

Effect of 3G cell phone exposure with computer controlled 2-D stepper motor on non-thermal activation of the hsp27/p38MAPK stress pathway in rat brain.

PubMed

Kesari, Kavindra Kumar; Meena, Ramovatar; Nirala, Jayprakash; Kumar, Jitender; Verma, H N

2014-03-01

Cell phone radiation exposure and its biological interaction is the present concern of debate. Present study aimed to investigate the effect of 3G cell phone exposure with computer controlled 2-D stepper motor on 45-day-old male Wistar rat brain. Animals were exposed for 2 h a day for 60 days by using mobile phone with angular movement up to zero to 30°. The variation of the motor is restricted to 90° with respect to the horizontal plane, moving at a pre-determined rate of 2° per minute. Immediately after 60 days of exposure, animals were scarified and numbers of parameters (DNA double-strand break, micronuclei, caspase 3, apoptosis, DNA fragmentation, expression of stress-responsive genes) were performed. Result shows that microwave radiation emitted from 3G mobile phone significantly induced DNA strand breaks in brain. Meanwhile a significant increase in micronuclei, caspase 3 and apoptosis were also observed in exposed group (P < 0.05). Western blotting result shows that 3G mobile phone exposure causes a transient increase in phosphorylation of hsp27, hsp70, and p38 mitogen-activated protein kinase (p38MAPK), which leads to mitochondrial dysfunction-mediated cytochrome c release and subsequent activation of caspases, involved in the process of radiation-induced apoptotic cell death. Study shows that the oxidative stress is the main factor which activates a variety of cellular signal transduction pathways, among them the hsp27/p38MAPK is the pathway of principle stress response. Results conclude that 3G mobile phone radiations affect the brain function and cause several neurological disorders.

Overexpression of Endogenous Anti-Oxidants with Selenium Supplementation Protects Trophoblast Cells from Reactive Oxygen Species-Induced Apoptosis in a Bcl-2-Dependent Manner.

PubMed

Khera, Alisha; Vanderlelie, Jessica J; Holland, Olivia; Perkins, Anthony V

2017-06-01

The human placenta provides life support for the developing foetus, and a healthy placenta is a prerequisite to a healthy start to life. Placental tissue is subject to oxidative stress which can lead to pathological conditions of pregnancy such as preeclampsia, preterm labour and intrauterine growth restriction. Up-regulation of endogenous anti-oxidants may alleviate placental oxidative stress and provide a therapy for these complications of pregnancy. In this study, selenium supplementation, as inorganic sodium selenite (NaSel) or organic selenomethionine (SeMet), was used to increase the protein production and cellular activity of the important redox active proteins glutathione peroxidase (GPx) and thioredoxin reductase (Thx-Red). Placental trophoblast cell lines, BeWo, JEG-3 and Swan-71, were cultured in various concentrations of NaSel or SeMet for 24 h and cell extracts prepared for western blots and enzyme assays. Rotenone and antimycin were used to stimulate mitochondrial reactive oxygen species (ROS) production and induce apoptosis. Trophoblast cells supplemented with 100 nM NaSel and 500 nM SeMet exhibited significantly enhanced expression and activity of both GPx and Thx-Red. Antimycin and rotenone were found to generate ROS when measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, and selenium supplementation was shown to reduce ROS production in a dose-dependent manner. Rotenone, 100 μM treatment for 4 h, caused trophoblast cell apoptosis as evidenced by increased Annexin V binding and decreased expression of Bcl-2. In both assays of apoptosis, selenium supplementation was able to prevent apoptosis, preserve Bcl-2 expression and protect trophoblast cells from mitochondrial oxidative stress. This data suggests that selenoproteins such as GPx and Thx-Red have an important role in protecting trophoblast cells from mitochondrial oxidative stress and that selenium supplementation may be important in treating some placental pathologies.

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout–derived cell line, RTG-2

USGS Publications Warehouse

Kurath, Gael; Purcell, Maureen K.; Wargo, Andrew; Park, Jeong Woo; Moon, Chang Hoon

2010-01-01

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout–derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H2O2 appear to increase chromatin bridges and resultant cytokinesis failure.

PubMed

Cho, Min-Guk; Ahn, Ju-Hyun; Choi, Hee-Song; Lee, Jae-Ho

2017-07-01

Aneuploidy, an abnormal number of chromosomes that is a hallmark of cancer cells, can arise from tetraploid/binucleated cells through a failure of cytokinesis. Reactive oxygen species (ROS) have been implicated in various diseases, including cancer. However, the nature and role of ROS in cytokinesis progression and related mechanisms has not been clearly elucidated. Here, using time-lapse analysis of asynchronously growing cells and immunocytochemical analyses of synchronized cells, we found that hydrogen peroxide (H 2 O 2 ) treatment at early mitosis (primarily prometaphase) significantly induced cytokinesis failure. Cytokinesis failure and the resultant formation of binucleated cells containing nucleoplasmic bridges (NPBs) seemed to be caused by increases in DNA double-strand breaks (DSBs) and subsequent unresolved chromatin bridges. We further found that H 2 O 2 induced mislocalization of Aurora B during mitosis. All of these effects were attenuated by pretreatment with N-acetyl-L-cysteine (NAC) or overexpression of Catalase. Surprisingly, the PARP inhibitor PJ34 also reduced H 2 O 2 -induced Aurora B mislocalization and binucleated cell formation. Results of parallel experiments with etoposide, a topoisomerase IIα inhibitor that triggers DNA DSBs, suggested that both DNA DSBs and Aurora B mislocalization contribute to chromatin bridge formation. Aurora B mislocalization also appeared to weaken the "abscission checkpoint". Finally, we showed that KRAS-induced binucleated cell formation appeared to be also H 2 O 2 -dependent. In conclusion, we propose that a ROS, mainly H 2 O 2 increases binucleation through unresolved chromatin bridges caused by DNA damage and mislocalization of Aurora B, the latter of which appears to augment the effect of DNA damage on chromatin bridge formation. Copyright © 2017 Elsevier Inc. All rights reserved.

Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210 leukemia cells by activation of caspase-3.

PubMed

de Mejia, Elvira Gonzalez; Wang, Wenyi; Dia, Vermont P

2010-03-01

Lunasin is a novel chemopreventive peptide featuring a cell adhesion motif composed of arginine-glycine-aspartate (RGD) which has been associated to cytotoxicity to established cell lines. The objectives of this study were to determine the effect of lunasin on the viability of L1210 leukemia cells and to understand the underlying mechanisms involved. Pure lunasin and lunasin enriched soy flour (LES) caused cytotoxicity to L1210 leukemia cells with IC(50) of 14 and 16 microM (lunasin equivalent), respectively. Simulated gastrointestinal digestion showed that 25% of the original amount of lunasin survived 3 h of pepsin digestion and 3% of lunasin remained after sequential pepsin-pancreatin digestion for a total of 6 h. Cell cycle analysis showed that lunasin caused a dose-dependent G2 cell cycle arrest and apoptosis. Treatment of L1210 leukemia cells with 1 mg/mL of LES for 18 h led to an increase in the amount of apoptotic cells from 2 to 40%. Compared to untreated cells, treatment with 1 mg/mL LES showed a 6-fold increase on the expressions of caspases-8 and -9, and and a 12-fold increase on the expression of caspase-3. These results showed for the first time that lunasin, a naturally occurring peptide containing an RGD motif, caused apoptosis to L1210 leukemia cells through caspase-3 activation.

Neuronal Sodium Channels in Neurodegeneration and Neuroprotection

DTIC Science & Technology

2002-06-01

following 2 h MCAo/ reperfusion injury Group’ Baselineb 2 h 4 h 6 h $ 24 1h Vehicle 37.0±0.7 38.3±0.7 37.1 ±0.8 37.5 ±0.7 36.6±0.8 RS (0.01 mg/kg) 36.4-±0.3...brain injury caused by middle cerebral neuronal cell death caused by ischemia results from artery occlusion (MCAo) for 2h followed by reperfusion a...expression following cerebral ischemia study, was delayed post- injury (i.e. > 2 -6h post- involves the up-regulation of several gene families injury ). This

Elicitation of anti-vesicular stomatitis virus cytotoxic T lymphocytes by using purified viral and cellular antigens incorporated into phospholipid vesicles.

PubMed

Ruebush, M J; Hale, A H; Harris, D T

1981-05-01

We evaluated the minimal molecular and cellular requirements for elicitation of anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). The results indicated that lipid vesicles containing the purified major surface glyco-protein of VSV (G protein) and purified H-2K(k) glycoproteins elicited specific H-2K(k)-restricted anti-VSV CTL. These antiviral CTL were shown to be Ly 1(-),2(+). However, both Ly 1(+),2(-) and Ly1(-),2(+) T-cell subpopulations were shown to be required for elicitation of these CTL.

Cytotoxicity and Induction of Inflammation by Pepsin in Acid in Bronchial Epithelial Cells

PubMed Central

Bathoorn, Erik; Daly, Paul; Gaiser, Birgit; Sternad, Karl; Poland, Craig; MacNee, William; Drost, Ellen M.

2011-01-01

Introduction. Gastroesophageal reflux has been associated with chronic inflammatory diseases and may be a cause of airway remodelling. Aspiration of gastric fluids may cause damage to airway epithelial cells, not only because acidity is toxic to bronchial epithelial cells, but also since it contains digestive enzymes, such as pepsin. Aim. To study whether pepsin enhances cytotoxicity and inflammation in airway epithelial cells, and whether this is pH-dependent. Methods. Human bronchial epithelial cells were exposed to increasing pepsin concentrations in varying acidic milieus, and cell proliferation and cytokine release were assessed. Results. Cell survival was decreased by pepsin exposure depending on its concentration (F = 17.4) and pH level of the medium (F = 6.5) (both P < 0.01). Pepsin-induced interleukin-8 release was greater at lower pH (F = 5.1; P < 0.01). Interleukin-6 induction by pepsin was greater at pH 1.5 compared to pH 2.5 (mean difference 434%; P = 0.03). Conclusion. Pepsin is cytotoxic to bronchial epithelial cells and induces inflammation in addition to acid alone, dependent on the level of acidity. Future studies should assess whether chronic aspiration causes airway remodelling in chronic inflammatory lung diseases. PMID:21785693

The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells.

PubMed

Jakab, Martin; Ketterl, Nina; Fürst, Johannes; Beyreis, Marlena; Kittl, Michael; Kiesslich, Tobias; Hauser-Kronberger, Cornelia; Gaisberger, Martin; Ritter, Markus

2017-01-01

Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICav comparable to 20 µM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨm within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨm within 24 h. We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion. © 2017 The Author(s). Published by S. Karger AG, Basel.

Substance P promotes the recovery of oxidative stress-damaged retinal pigmented epithelial cells by modulating Akt/GSK-3β signaling.

PubMed

Baek, Sang-Min; Yu, Seung-Young; Son, Youngsook; Hong, Hyun Sook

2016-01-01

Senescence of the retina causes an accumulation of reactive oxygen species (ROS). Oxidative stress associated with ROS can damage RPE cells, leading to neovascularization and severe ocular disorders, including age-related macular degeneration (AMD). Thus, the early treatment of the damage caused by oxidative stress is critical for preventing the development of ocular diseases such as AMD. In this study, we examined the role of substance P (SP) in the recovery of RPE cells damaged by oxidative stress. To induce oxidative stress, RPE cells were treated with H2O2 at various doses. Recovery from oxidative stress was studied following treatment with SP by analyzing cell viability, cell proliferation, cell apoptosis, and Akt/glycogen synthase kinase (GSK)-3β activation in RPE cells in vitro. H2O2 treatment reduced cellular viability in a dose-dependent manner. SP inhibited the reduction of cell viability due to H2O2 and caused increased cell proliferation and decreased cell apoptosis. Cell survival under oxidative stress requires the activation of Akt signaling that enables cells to resist oxidative stress-induced damage. SP treatment activated Akt/GSK-3β signaling in RPE cells, which were damaged due to oxidative stress, and the inhibition of Akt signaling in SP-treated RPE cells prevented SP-induced recovery. Pretreatment with the neurokinin 1 receptor (NK1R) antagonist reduced the recovery effect of SP on damaged RPE cells. SP can protect RPE cells from oxidant-induced cell death by activating Akt/GSK-3β signaling via NK1R. This study suggests the possibility of SP as a treatment for oxidative stress-related diseases.

Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.

PubMed

Gregory, Jesse F; DeRatt, Barbara N; Rios-Avila, Luisa; Ralat, Maria; Stacpoole, Peter W

2016-07-01

The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine → cystathionine → cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 μmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Extractive Fermentation of Lactic Acid in Lactic Acid Bacteria Cultivation: A Review.

PubMed

Othman, Majdiah; Ariff, Arbakariya B; Rios-Solis, Leonardo; Halim, Murni

2017-01-01

Lactic acid bacteria are industrially important microorganisms recognized for their fermentative ability mostly in their probiotic benefits as well as lactic acid production for various applications. Nevertheless, lactic acid fermentation often suffers end-product inhibition which decreases the cell growth rate. The inhibition of lactic acid is due to the solubility of the undissociated lactic acid within the cytoplasmic membrane and insolubility of dissociated lactate, which causes acidification of cytoplasm and failure of proton motive forces. This phenomenon influences the transmembrane pH gradient and decreases the amount of energy available for cell growth. In general, the restriction imposed by lactic acid on its fermentation can be avoided by extractive fermentation techniques, which can also be exploited for product recovery.

Extractive Fermentation of Lactic Acid in Lactic Acid Bacteria Cultivation: A Review

PubMed Central

Othman, Majdiah; Ariff, Arbakariya B.; Rios-Solis, Leonardo; Halim, Murni

2017-01-01

Lactic acid bacteria are industrially important microorganisms recognized for their fermentative ability mostly in their probiotic benefits as well as lactic acid production for various applications. Nevertheless, lactic acid fermentation often suffers end-product inhibition which decreases the cell growth rate. The inhibition of lactic acid is due to the solubility of the undissociated lactic acid within the cytoplasmic membrane and insolubility of dissociated lactate, which causes acidification of cytoplasm and failure of proton motive forces. This phenomenon influences the transmembrane pH gradient and decreases the amount of energy available for cell growth. In general, the restriction imposed by lactic acid on its fermentation can be avoided by extractive fermentation techniques, which can also be exploited for product recovery. PMID:29209295

Hypoxia promotes luteal cell death in bovine corpus luteum.

PubMed

Nishimura, Ryo; Komiyama, Junichi; Tasaki, Yukari; Acosta, Tomas J; Okuda, Kiyoshi

2008-03-01

Low oxygen caused by a decreasing blood supply is known to induce various responses of cells, including apoptosis. The present study was conducted to examine whether low-oxygen conditions (hypoxia) induce luteal cell apoptosis in cattle. Bovine midluteal cells incubated under hypoxia (3% O(2)) showed significantly more cell death than did those incubated under normoxia (20% O(2)) at 24 and 48 h of culture, and had significantly lower progesterone (P4) levels starting at 8 h. Characteristic features of apoptosis, such as shrunken nuclei and DNA fragmentation, were observed in cells cultured under hypoxia for 48 h. Hypoxia increased the mRNA expressions of BNIP3 and caspase 3 at 24 and 48 h of culture. Hypoxia had no significant effect on the expressions of BCL2 and BAX mRNA. Hypoxia also increased BNIP3 protein, and activated caspase-3. Treatment of P4 attenuated cell death, caspase-3 mRNA expression, and caspase-3 activity under hypoxia. Overall results of the present study indicate that hypoxia induces luteal cell apoptosis by enhancing the expression of proapoptotic protein, BNIP3, and by activating caspase-3, and that the induction of apoptosis by hypoxia is partially caused by a decrease in P4 production. Because hypoxia suppresses P4 synthesis in bovine luteal cells, we suggest that oxygen deficiency caused by a decreasing blood supply in bovine corpus luteum is one of the major factors contributing to both functional and structural luteolysis.

Influence of chlorine or fluorine substitution on the estrogenic properties of 1-alkyl-2,3,5-tris(4-hydroxyphenyl)-1H-pyrroles.

PubMed

Schäfer, Anja; Wellner, Anja; Strauss, Martin; Schäfer, Andreas; Wolber, Gerhard; Gust, Ronald

2012-11-26

In continuation of our previous work, several 1-alkyl-2,3,5-tris(4-hydroxyphenyl)aryl-1H-pyrroles with chlorine or fluorine substituents in the aryl residues were synthesized and tested for estrogen receptor (ER) binding at isolated ERα/ERβ receptors (HAP assay) and in transactivation assays using ERα-positive MCF-7/2a as well as U2-OS/ERα and U2-OS/ERβ cells. In the competition experiment at ERα the compounds displayed very high relative binding affinities of up to 37% (determined for 8m) but with restricted subtype selectivity (e.g., ERα/ERβ (8m) = 9). The highest estrogenic potency in ERα-positive MCF-7/2a cells was determined for 2,3,5-tris(2-fluoro-4-hydroxyphenyl)-1-propyl-1H-pyrrole 8m (EC(50) = 23 nM), while in U2-OS/ERα cells 2-(2-fluoro-4-hydroxyphenyl)-3,5-bis(4-hydroxyphenyl)-1-propyl-1H-pyrrole 8b (EC(50) = 0.12 nM) was the most potent agonist, only 30-fold less active than estradiol (E2, EC(50) = 0.004 nM). In U2-OS/ERβ cells for all pyrroles no transactivation could be observed, which indicates that they are selective ERα agonists in cellular systems.

Stimulus-Responsive Plasmonic Chiral Signals of Gold Nanorods Organized on DNA Origami.

PubMed

Jiang, Qiao; Liu, Qing; Shi, Yuefeng; Wang, Zhen-Gang; Zhan, Pengfei; Liu, Jianbing; Liu, Chao; Wang, Hui; Shi, Xinghua; Zhang, Li; Sun, Jiashu; Ding, Baoquan; Liu, Minghua

2017-11-08

In response to environmental variations, living cells need to arrange the conformational changes of macromolecules to achieve the specific biofunctions. Inspired by natural molecular machines, artificial macromolecular assemblies with controllable nanostructures and environmentally responsive functions can be designed. By assembling macromolecular nanostructures with noble metal nanoparticles, environmental information could be significantly amplified and modulated. However, manufacturing dynamic plasmonic nanostructures that are efficiently responsive to different stimuli is still a challenging task. Here we demonstrate a stimulus-responsive plasmonic nanosystem based on DNA origami-organized gold nanorods (GNRs). L-shaped GNR dimers were assembled on rhombus-shaped DNA origami templates. The geometry and chiral signals of the GNR nanoarchitectures respond to multiple stimuli, including glutathione reduction, restriction enzyme action, pH change, or photoirradiation. While the glutathione reduction or restriction enzyme caused irreversible changes in the plasmonic circular dichroism (CD) signals, both pH and light irradiation triggered reversible changes in the plasmonic CD. Our system transduces external stimuli into conformational changes and circular dichroism responses in near-infrared (NIR) wavelengths. By this approach, programmable optical reporters for essential biological signals can be fabricated.

Activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress.

PubMed

Lu, Jun; Chen, Jian; Xu, Nianjun; Wu, Jun; Kang, Yani; Shen, Tingting; Kong, Hualei; Ma, Chao; Cheng, Ming; Shao, Zhifeng; Xu, Ling; Zhao, Xiaodong

2016-09-06

Application of cisplatin (DDP) for treating lung cancer is restricted due to its toxicity and lung cancer's drug resistance. In this study, we examined the effect of Jinfukang (JFK), an effective herbal medicine against lung cancer, on DDP-induced cytotoxicity in lung cancer cells. Morphologically, we observed that JFK increases DDP-induced pro-apoptosis in A549 cells in a synergistic manner. Transcriptome profiling analysis indicated that the combination of JFK and DDP regulates genes involved in apoptosis-related signaling pathways. Moreover, we found that the combination of JFK and DDP produces synergistic pro-apoptosis effect in other lung cancer cell lines, such as NCI-H1975, NCI-H1650, and NCI-H2228. Particularly, we demonstrated that AIFM2 is activated by the combined treatment of JFK and DDP and partially mediates the synergistic pro-apoptosis effect. Collectively, this study not only offered the first evidence that JFK promotes DDP-induced cytotoxicity, and activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress, but also provided a novel insight for improving cytotoxicity by combining JFK with DDP to treat lung cancer cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozawa, H.; Iwaguchi, T.; Kataoka, T.

1987-12-01

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurredmore » between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the effector phase as well as in the induction phase. The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the bystander effect.« less

Multiple discrete encephalitogenic epitopes of the autoantigen myelin basic protein include a determinant for I-E class II-restricted T cells

PubMed Central

1988-01-01

Immunization with the autoantigen myelin basic protein (MBP) causes experimental allergic encephalomyelitis (EAE). Initial investigations indicated that encephalitogenic murine determinants of MBP were located only within MBP 1-37 and MBP 89-169. Encephalitogenic T cell epitopes within these fragments have been identified. Each epitope is recognized by T cells in association with separate allelic I-A molecules. A hybrid I-E-restricted T cell clone that recognizes intact mouse (self) MBP has been examined. The epitope recognized by this clone includes MBP residues 35-47. When tested in vivo, p35-47 causes EAE. T cell recognition of p35-47 occurs only in association with I-E molecules. These results provide the first clear example that antigen-specific T cells restricted by I-E class II molecules participate in murine autoimmune disease. Furthermore, it is clear that there are multiple (at least three) discrete encephalitogenic T cell epitopes of this autoantigen, each recognized in association with separate allelic class II molecules. These results may be relevant to human autoimmune diseases whose susceptibility is associated with more than one HLA-D molecule. PMID:2459291

The NRF2 Activation and Antioxidative Response Are Not Impaired Overall during Hyperoxia-Induced Lung Epithelial Cell Death

PubMed Central

Potteti, Haranatha R.; Reddy, Narsa M.; Hei, Tom K.; Kalvakolanu, Dhananjaya V.; Reddy, Sekhar P.

2013-01-01

Lung epithelial and endothelial cell death caused by pro-oxidant insults is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients. The NF-E2-related factor 2 (NRF2) activation in response to oxidant exposure is crucial to the induction of several antioxidative and cytoprotective enzymes that mitigate cellular stress. Since prolonged exposure to hyperoxia causes cell death, we hypothesized that chronic hyperoxia impairs NRF2 activation, resulting in cell death. To test this hypothesis, we exposed nonmalignant small airway epithelial cells (AECs) to acute (1–12 h) and chronic (36–48 h) hyperoxia and evaluated cell death, NRF2 nuclear accumulation and target gene expression, and NRF2 recruitment to the endogenous HMOX1 and NQO1 promoters. As expected, hyperoxia gradually induced death in AECs, noticeably and significantly by 36 h; ~60% of cells were dead by 48 h. However, we unexpectedly found increased expression levels of NRF2-regulated antioxidative genes and nuclear NRF2 in AECs exposed to chronic hyperoxia as compared to acute hyperoxia. Chromatin Immunoprecipitation (ChIP) assays revealed an increased recruitment of NRF2 to the endogenous HMOX1 and NQO1 promoters in AECs exposed to acute or chronic hyperoxia. Thus, our findings demonstrate that NRF2 activation and antioxidant gene expression are functional during hyperoxia-induced lung epithelial cell death and that chronic hyperoxia does not impair NRF2 signaling overall. PMID:23738042

Major Histocompatibilty Complex-Restricted Adaptive Immune Responses to CT26 Colon Cancer Cell Line in Mixed Allogeneic Chimera.

PubMed

Lee, K W; Choi, B; Kim, Y M; Cho, C W; Park, H; Moon, J I; Choi, G-S; Park, J B; Kim, S J

2017-06-01

Although the induction of mixed allogeneic chimera shows promising clinical tolerance results in organ transplantation, its clinical relevance as an anti-cancer therapy is yet unknown. We introduced a mixed allogenic chimera setting with the use of a murine colon cancer cell line, CT26, by performing double bone marrow transplantation. We analyzed donor- and recipient-restricted anti-cancer T-cell responses, and phenotypes of subpopulations of T cells. The protocol involves challenging 1 × 10 5 cells of CT26 cells intra-hepatically on day 50 after bone marrow transplantation, and, by use of CT26 lysates and an H-2L d -restricted AH1 pentamer, flow cytometric analysis was performed to detect the generation of cancer-specific CD4 + and CD8 + T cells at various time points. We found that immunocompetence against tumors depends heavily on cancer-specific CD8 + T-cell responses in a major histocompatibility complex-restricted manner; the evidence was further supported by the increase of interferon-γ-secreting CD4 + T cells. Moreover, we demonstrated that during the effector immune response to CT26 cancer challenge, there was a presence of central memory cells (CD62L hi CCR7 + ) as well as effector memory cells (CD62L lo CCR7 - ). Moreover, mixed allogeneic chimeras (BALB/c to C56BL/6 or vice versa) showed similar or heightened immune responses to CT26 cells compared with that of wild-type mice. Our results suggest that the responses of primary immunocompetency and of pre-existing memory T cells against allogeneic cancer are sustained and preserved long-term in a mixed allogeneic chimeric environment. Copyright © 2017 Elsevier Inc. All rights reserved.

IDH1(R132H) mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation status.

PubMed

Kessler, Jacqueline; Güttler, Antje; Wichmann, Henri; Rot, Swetlana; Kappler, Matthias; Bache, Matthias; Vordermark, Dirk

2015-09-01

In malignant glioma the presence of the IDH1 mutation (IDH1(R132H)) is associated with better clinical outcome. However, it is unclear whether IDH1 mutation is associated with a less aggressive phenotype or directly linked to increased sensitivity to radiotherapy. We determined the influence of IDH1(R132H) mutant protein on proliferation and growth in 3D culture, migration, cell survival and radiosensitivity in vitro under normoxia (21% O2) and hypoxia (<1% O2) in a panel of human malignant glioma cell lines (U-251MG, U-343MG, LN-229) with stable overexpression of wild-type (IDH1(wt)) and mutated IDH1 (IDH1(R132H)). Overexpression of IDH1(R132H) in glioma cells resulted in slightly decreased cell proliferation, considerably reduced cell migration and caused differences in growth properties in 3D spheroid cultures. Furthermore, IDH1(R132H)-positive cells consistently demonstrated an increased radiosensitivity in human malignant glioma cells U-251MG (DMF10: 1.52, p<0.01 and 1.42, p<0.01), U-343MG (DMF10: 1.78, p<0.01 and 1.75, p<0.01) and LN-229 (DMF10: 1.41, p<0.05 and 1.68, p<0.01) under normoxia and hypoxia, respectively. Our data indicate that IDH1(R132H) mutation causes both a less aggressive biological behavior and direct radiosensitization of human malignant glioma cells. Targeting IDH1 appears to be an attractive approach in combination with radiotherapy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Retinal damage profiles and neuronal effects of laser treatment: comparison of a conventional photocoagulator and a novel 3-nanosecond pulse laser.

PubMed

Wood, John P M; Shibeeb, O'Sam; Plunkett, Malcolm; Casson, Robert J; Chidlow, Glyn

2013-03-28

To determine detailed effects to retinal cells and, in particular, neurons following laser photocoagulation using a conventional 532 nm Nd:YAG continuous wave (CW) laser. Furthermore, to determine whether a novel 3 ns pulse laser (retinal regeneration therapy; 2RT) could specifically ablate retinal pigment epithelium (RPE) cells without causing collateral damage to other retinal cells. Adult Dark Agouti (DA) rats were separated into four groups: control, CW laser (12.7 J/cm(2)/pulse, 100 ms pulse duration), or 3 ns pulse 2RT laser at one of two energy settings ("High," 2RT-H, 163 mJ/cm(2)/pulse; "Low," 2RT-L, 109 mJ/cm(2)/pulse). Animals were treated and killed after 6 hours to 7 days, and retina/RPE was analyzed by histologic assessment, Western blot, polymerase chain reaction, and immunohistochemistry. Both lasers caused focal loss of RPE cells with no destruction of Bruch's membrane; RPE cells were present at lesion sites again within 7 days of treatments. CW and 2RT-H treatments caused extensive and moderate damage, respectively, to the outer retina. There were no obvious effects to horizontal, amacrine, or ganglion cells, as defined by immunolabeling, but an activation of PKCα within bipolar cells was noted. There was little discernible damage to any cells other than the RPE with the 2RT-L treatment. Conventional laser photocoagulation caused death of RPE cells with associated widespread damage to the outer retina but little influence on the inner retina. The novel 3 ns 2RT laser, however, was able to selectively kill RPE cells without causing collateral damage to photoreceptors. Potential benefits of this laser for clinical treatment of diabetic macular edema are discussed.

Development and characterization of a novel human Waldenström Macroglobulinemia cell line (RPCI-WM1; Roswell Park Cancer Institute-Waldenström Macroglobulinemia 1)

PubMed Central

Chitta, Kasyapa S.; Paulus, Aneel; Ailawadhi, Sikander; Foster, Barbara A.; Moser, Michael T.; Starostik, Petr; Masood, Aisha; Sher, Taimur; Miller, Kena C.; Iancu, Dan M.; Conroy, Jeffrey; Nowak, Norma J.; Sait, Sheila N.; Personett, David A.; Coleman, Morton; Furman, Richard R.; Martin, Peter; Ansell, Stephen M.; Lee, Kelvin; Chanan-Khan, Asher A.

2015-01-01

Understanding the biology of Waldenström Macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secreted human IgM (hIgM) with k-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2) with a consistent amplification of 14q32 (IgH) identical to its founding tumor sample. Clonal relationship was confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Over all, RPCI-WM1 represents a valuable model to study WM. PMID:22812491

Development and characterization of a novel human Waldenström macroglobulinemia cell line: RPCI-WM1, Roswell Park Cancer Institute - Waldenström Macroglobulinemia 1.

PubMed

Chitta, Kasyapa S; Paulus, Aneel; Ailawadhi, Sikander; Foster, Barbara A; Moser, Michael T; Starostik, Petr; Masood, Aisha; Sher, Taimur; Miller, Kena C; Iancu, Dan M; Conroy, Jeffrey; Nowak, Norma J; Sait, Sheila N; Personett, David A; Coleman, Morton; Furman, Richard R; Martin, Peter; Ansell, Stephen M; Lee, Kelvin; Chanan-Khan, Asher A

2013-02-01

Understanding the biology of Waldenström macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secretes human immunoglobulin M (h-IgM) with κ-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2), with a consistent amplification of 14q32 (immunoglobulin heavy chain; IgH) identical to its founding tumor sample. The clonal relationship is confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in the MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Overall, RPCI-WM1 represents a valuable model to study Waldenström macroglobulinemia.

One-step synthesis, toxicity assessment and degradation in tumoral pH environment of SiO2@Ag core/shell nanoparticles

NASA Astrophysics Data System (ADS)

De Matteis, Valeria; Rizzello, Loris; Di Bello, Maria Pia; Rinaldi, Rosaria

2017-06-01

The unique physicochemical properties of SiO2@Ag core/shell nanoparticles make them a promising tool in nanomedicine, where they are used as nanocarriers for several biomedical applications, including (but not restricted to) cancer treatment. However, a comprehensive estimation of their potential toxicity, as well as their degradation in the tumor microenvironment, has not been extensively addressed yet. We investigated in vitro the viability, the reactive oxygen species (ROS) production, the DNA damage level, and the nanoparticle uptake on HeLa cells, used as model cancer cells. In addition, we studied the NPs degradation profile at pH 6.5, to mimic the tumor microenvironment, and at the neutral and physiological (pH 7-7.4). Our experiments demonstrate that the silver shell dissolution is promoted under acidic conditions, which could be related to cell death induction. Our evidences demonstrate that SiO2@Ag nanoparticles possess the ability of combining an effective cancer cell treatment (through local silver ions release) together with a possible controlled release of bioactive compounds encapsulated in the silica as future application.

Molecular Epidemiology of Adenovirus Type 7 in the United States, 1966–20001

PubMed Central

Xu, Wanhong; Gerber, Susan I.; Gray, Gregory C.; Schnurr, David; Kajon, Adriana E.; Anderson, Larry J.

2002-01-01

Genetic variation among 166 isolates of human adenovirus 7 (Ad7) obtained from 1966 to 2000 from the United States and Eastern Ontario, Canada, was determined by genome restriction analysis. Most (65%) isolates were identified as Ad7b. Two genome types previously undocumented in North America were also identified: Ad7d2 (28%), which first appeared in 1993 and was later identified throughout the Midwest and Northeast of the United States and in Canada; and Ad7h (2%), which was identified only in the U.S. Southwest in 1998 and 2000. Since 1996, Ad7d2 has been responsible for several civilian outbreaks of Ad7 disease and was the primary cause of a large outbreak of respiratory illness at a military recruit training center. The appearance of Ad7d2 and Ad7h in North America represents recent introduction of these viruses from previously geographically restricted areas and may herald a shift in predominant genome type circulating in the United States. PMID:11927024

IFITM Proteins Restrict Viral Membrane Hemifusion

PubMed Central

Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

2013-01-01

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection. PMID:23358889

Brief Report: Late-Onset Cryopyrin-Associated Periodic Syndrome Due to Myeloid-Restricted Somatic NLRP3 Mosaicism.

PubMed

Mensa-Vilaro, Anna; Teresa Bosque, María; Magri, Giuliana; Honda, Yoshitaka; Martínez-Banaclocha, Helios; Casorran-Berges, Marta; Sintes, Jordi; González-Roca, Eva; Ruiz-Ortiz, Estibaliz; Heike, Toshio; Martínez-Garcia, Juan J; Baroja-Mazo, Alberto; Cerutti, Andrea; Nishikomori, Ryuta; Yagüe, Jordi; Pelegrín, Pablo; Delgado-Beltran, Concha; Aróstegui, Juan I

2016-12-01

Gain-of-function NLRP3 mutations cause cryopyrin-associated periodic syndrome (CAPS), with gene mosaicism playing a relevant role in the pathogenesis. This study was undertaken to characterize the genetic cause underlying late-onset but otherwise typical CAPS. We studied a 64-year-old patient who presented with recurrent episodes of urticaria-like rash, fever, conjunctivitis, and oligoarthritis at age 56 years. DNA was extracted from both unfractionated blood and isolated leukocyte and CD34+ subpopulations. Genetic studies were performed using both the Sanger method of DNA sequencing and next-generation sequencing (NGS) methods. In vitro and ex vivo analyses were performed to determine the consequences that the presence of the variant have in the normal structure or function of the protein of the detected variant. NGS analyses revealed the novel p.Gln636Glu NLRP3 variant in unfractionated blood, with an allele frequency (18.4%) compatible with gene mosaicism. Sanger sequence chromatograms revealed a small peak corresponding to the variant allele. Amplicon-based deep sequencing revealed somatic NLRP3 mosaicism restricted to myeloid cells (31.8% in monocytes, 24.6% in neutrophils, and 11.2% in circulating CD34+ common myeloid progenitor cells) and its complete absence in lymphoid cells. Functional analyses confirmed the gain-of-function behavior of the gene variant and hyperactivity of the NLRP3 inflammasome in the patient. Treatment with anakinra resulted in good control of the disease. We identified the novel gain-of-function p.Gln636Glu NLRP3 mutation, which was detected as a somatic mutation restricted to myeloid cells, as the cause of late-onset but otherwise typical CAPS. Our results expand the diversity of CAPS toward milder phenotypes than previously reported, including those starting during adulthood. © 2016, American College of Rheumatology.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Chao; Yang, Bo; Yang, Zhi

Highlights: Black-Right-Pointing-Pointer UVB radiated skin keratinocytes show cyclophilin D (Cyp-D) upregulation. Black-Right-Pointing-Pointer NAC inhibits UVB induced Cyp-D expression, while H{sub 2}O{sub 2} facilitates it. Black-Right-Pointing-Pointer Cyp-D-deficient cells are significantly less susceptible to UVB induced cell death. Black-Right-Pointing-Pointer Over-expression of Cyp-D causes spontaneous keratinocytes cell death. -- Abstract: UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaTmore » cell line demonstrated that UVB radiation and hydrogen peroxide (H{sub 2}O{sub 2}) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H{sub 2}O{sub 2}-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H{sub 2}O{sub 2}-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.« less

The effect of bovine BST2A1 on the release and cell-to-cell transmission of retroviruses.

PubMed

Liang, Zhibin; Zhang, Yang; Song, Jie; Zhang, Hui; Zhang, Suzhen; Li, Yue; Tan, Juan; Qiao, Wentao

2017-09-06

Human BST2 (hBST2, also called Tetherin) is a host restriction factor that blocks the release of various enveloped viruses. BST2s from different mammals also possess antiviral activity. Bovine BST2s (bBST2s), bBST2A1 and bBST2A2, reduce production of cell-free bovine leukemia virus (BLV) and vesicular stomatitis virus (VSV). However, the effect of bBST2 on other retroviruses remains unstudied. Here, we studied the antiviral activity of wildtype and mutant bBST2A1 proteins on retroviruses including human immunodeficiency virus type 1 (HIV-1), prototypic foamy virus (PFV), bovine foamy virus (BFV) and bovine immunodeficiency virus (BIV). The results showed that wildtype bBST2A1 suppressed the release of HIV-1, PFV and BFV. We also generated bBST2A1 mutants, and found that GPI anchor and dimerization, but not glycosylation, are essential for antiviral activity of bBST2A1. Moreover, unlike hBST2, bBST2A1 displayed no inhibitory effect on cell-to-cell transmission of PFV, BFV and BIV. Our data suggested that bBST2A1 inhibited retrovirus release, however, had no effect on cell-to-cell transmission of retroviruses.

Vaccine and Monoclonal Antibody That Enhance Mouse Resistance to Candidiasis ▿

PubMed Central

Xin, Hong; Cutler, Jim E.

2011-01-01

Previously we showed that antibodies specific for the glycan β-1,2-mannotriose [β-(Man)3] on the cell surface of Candida albicans protect mice against disseminated candidiasis (H. Xin, S. Dziadek, D. R. Bundle, and J. E. Cutler, Proc. Natl. Acad. Sci. U. S. A. 105:13526–13531, 2008). Furthermore, six 14-mer peptides that are within the N-terminal portion of C. albicans wall proteins were conjugated to the glycan in an attempt to create immunogenic glycopeptide conjugates. By a dendritic cell (DC)-based immunization approach, all were immunogenic and three of the six conjugates induced a high degree of protection in mice. Interestingly, whereas all six peptides induced antibody responses when used alone to pulse DCs for subsequent immunizations, three peptides induced protection, and one in particular, peptide Fba (derived from fructose-bisphosphate aldolase), induced robust protective responses and is the focus of the current work. Fba peptide is not restricted by the major histocompatibility complex class II (MHC-II), as it induced anti-Fba antibodies in mice of different H-2 haplotypes and in rabbits. Furthermore, the peptide induced protection against disease caused by different C. albicans strains. Partial protection was achieved when alum was used in place of DCs for Fba immunizations. The passive transfer of immune sera from Fba-vaccinated mice, but not immune serum preabsorbed with fungal cells, conferred protection in naïve mice. This result, along with our finding that a monoclonal antibody specific for the peptide, E2-9 (IgM), protected mice against candidiasis, provide strong evidence that antibodies contribute to protection. Our work demonstrates the utility of cell wall peptides alone or as glycopeptides in vaccines designed for the induction of immunity against candidiasis and monoclonal antibodies as a rapid immunoprotective approach against the disease. PMID:21832099

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts.

PubMed

Inturi, Swetha; Tewari-Singh, Neera; Gu, Mallikarjuna; Shrotriya, Sangeeta; Gomez, Joe; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

2011-12-15

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities. Copyright © 2011 Elsevier Inc. All rights reserved.

Anti-bacterial immunity to Listeria monocytogenes in allogeneic bone marrow chimera in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onoe, K.; Good, R.A.; Yamamoto, K.

1986-06-01

Protection and delayed-type hypersensitivity (DTH) to the facultative intracellular bacterium Listeria monocytogenes (L.m.) were studied in allogeneic and syngeneic bone marrow chimeras. Lethally irradiated AKR (H-2k) mice were successfully reconstituted with marrow cells from C57BL/10 (B10) (H-2b), B10 H-2-recombinant strains or syngeneic mice. Irradiated AKR mice reconstituted with marrow cells from H-2-compatible B10.BR mice, (BR----AKR), as well as syngeneic marrow cells, (AKR----AKR), showed a normal level of responsiveness to the challenge stimulation with the listeria antigens when DTH was evaluated by footpad reactions. These mice also showed vigorous activities in acquired resistance to the L.m. By contrast, chimeric mice thatmore » had total or partial histoincompatibility at the H-2 determinants between donor and recipient, (B10----AKR), (B10.AQR----AKR), (B10.A(4R)----AKR), or (B10.A(5R)----AKR), were almost completely unresponsive in DTH and antibacterial immunity. However, when (B10----AKR) H-2-incompatible chimeras had been immunized with killed L.m. before challenge with live L.m., these mice manifested considerable DTH and resistance to L.m. These observations suggest that compatibility at the entire MHC between donor and recipient is required for bone marrow chimeras to be able to manifest DTH and protection against L.m. after a short-term immunization schedule. However, this requirement is overcome by a preceding or more prolonged period of immunization with L.m. antigens. These antigens, together with marrow-derived antigen-presenting cells, can then stimulate and expand cell populations that are restricted to the MHC (H-2) products of the donor type.« less

Dynamics of keratinocytes in vivo using HO labeling: a sensitive marker of epidermal proliferation state.

PubMed

Hsieh, Elaine A; Chai, Christine M; de Lumen, Benito O; Neese, Richard A; Hellerstein, Marc K

2004-09-01

A heavy water ((2)H(2)O) labeling method recently developed to measure cell proliferation in vivo is applied here to the measurement of murine epidermal cell turnover and to investigate conditions in which keratinocyte proliferation is either inhibited or stimulated. The technique is based on incorporation of (2)H(2)O into the deoxyribose moiety of deoxyribonucleotides in dividing cells. Label incorporation and die-away studies in cells isolated from C57BL/6J mouse epidermis revealed the replacement rate to be 34%-44% per wk (half-life of 1.6-2 wk). The kinetics provided evidence of a non-proliferative subpopulation of cells (10%-15% of the total) within the epidermis. Topical administration of 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate for 3 wk increased epidermal cell proliferation by 55% in SENCAR mice. Topical addition of lunasin, an anti-mitotic agent from soy, decreased epidermal cell proliferation modestly though significantly (16% given alone, 9% given with carcinogens). Caloric restriction (by 33% of energy intake) for 4 wk decreased the epidermal cell proliferation rate by 45% in C57BL/6J mice. In summary, epidermal cell proliferation can be measured in vivo using (2)H(2)O labeling in normal, hyper- and hypo-proliferative conditions. Potential applications of this inherently safe method in humans might include studies of psoriasis, wound healing, chemopreventive agents, and caloric intake.

The hERG K+ channel: target and antitarget strategies in drug development.

PubMed

Raschi, Emanuel; Vasina, Valentina; Poluzzi, Elisabetta; De Ponti, Fabrizio

2008-03-01

The human ether-à-go-go related gene (hERG) K+ channel is of great interest for both basic researchers and clinicians because its blockade by drugs can lead to QT prolongation, which is a risk factor for torsades de pointes, a potentially life-threatening arrhythmia. A growing list of agents with "QT liability" have been withdrawn from the market or restricted in their use, whereas others did not even receive regulatory approval for this reason. Thus, hERG K+ channels have become a primary antitarget (i.e. an unwanted target) in drug development because their blockade causes potentially serious side effects. On the other hand, the recent identification and functional characterization of hERG K+ channels not only in the heart, but also in several other tissues (e.g. neurons, smooth muscle and cancer cells) may have far reaching implications for drug development for a possible exploitation of hERG as a target, especially in oncology and cardiology.

Mapping immune cell infiltration using restricted diffusion MRI.

PubMed

Yeh, Fang-Cheng; Liu, Li; Hitchens, T Kevin; Wu, Yijen L

2017-02-01

Diffusion MRI provides a noninvasive way to assess tissue microstructure. Based on diffusion MRI, we propose a model-free method called restricted diffusion imaging (RDI) to quantify restricted diffusion and correlate it with cellularity. An analytical relation between q-space signals and the density of restricted spins was derived to quantify restricted diffusion. A phantom study was conducted to investigate the performance of RDI, and RDI was applied to an animal study to assess immune cell infiltration in myocardial tissues with ischemia-reperfusion injury. Our phantom study showed a correlation coefficient of 0.998 between cell density and the restricted diffusion quantified by RDI. The animal study also showed that the high-value regions in RDI matched well with the macrophage infiltration areas in the H&E stained slides. In comparison with diffusion tensor imaging (DTI), RDI exhibited its outperformance to detect macrophage infiltration and delineate inflammatory myocardium. RDI can be used to reveal cell density and detect immune cell infiltration. RDI exhibits better specificity than the diffusivity measurement derived from DTI. Magn Reson Med 77:603-612, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Identification of a G protein coupled receptor induced in activated T cells.

PubMed

Kaplan, M H; Smith, D I; Sundick, R S

1993-07-15

Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

Inhibition of lipolysis in the novel transgenic quail model overexpressing G0/G1 switch gene 2 in the adipose tissue during feed restriction.

PubMed

Shin, Sangsu; Choi, Young Min; Han, Jae Yong; Lee, Kichoon

2014-01-01

In addition to the issue of obesity in humans, the production of low-fat meat from domestic animals is important in the agricultural industry to satisfy consumer demand. Understanding the regulation of lipolysis in adipose tissue could advance our knowledge to potentially solve both issues. Although the G0/G1 switch gene 2 (G0S2) was recently identified as an inhibitor of adipose triglyceride lipase (ATGL) in vitro, its role in vivo has not been fully clarified. This study was conducted to investigate the role of G0S2 gene in vivo by using two independent transgenic quail lines during different energy conditions. Unexpectedly, G0S2 overexpression had a negligible effect on plasma NEFA concentration, fat cell size and fat pad weight under ad libitum feeding condition when adipose lipolytic activity is minimal. A two-week feed restriction in non-transgenic quail expectedly caused increased plasma NEFA concentration and dramatically reduced fat cell size and fat pad weight. Contrary, G0S2 overexpression under a feed restriction resulted in a significantly less elevation of plasma NEFA concentration and smaller reductions in fat pad weights and fat cell size compared to non-transgenic quail, demonstrating inhibition of lipolysis and resistance to loss of fat by G0S2. Excessive G0S2 inhibits lipolysis in vivo during active lipolytic conditions, such as food restriction and fasting, suggesting G0S2 as a potential target for treatment of obesity. In addition, transgenic quail are novel models for studying lipid metabolism and mechanisms of obesity.

Hydrogen sulphide facilitates exocytosis by regulating the handling of intracellular calcium by chromaffin cells.

PubMed

de Pascual, Ricardo; Baraibar, Andrés M; Méndez-López, Iago; Pérez-Ciria, Martín; Polo-Vaquero, Ignacio; Gandía, Luis; Ohia, Sunny E; García, Antonio G; de Diego, Antonio M G

2018-05-02

Gasotransmitter hydrogen sulphide (H 2 S) has emerged as a regulator of multiple physiological and pathophysiological processes throughout. Here, we have investigated the effects of NaHS (fast donor of H 2 S) and GYY4137 (GYY, slow donor of H 2 S) on the exocytotic release of catecholamines from fast-perifused bovine adrenal chromaffin cells (BCCs) challenged with sequential intermittent pulses of a K + -depolarizing solution. Both donors caused a concentration-dependent facilitation of secretion. This was not due to an augmentation of Ca 2+ entry through voltage-activated Ca 2+ channels (VACCs) because, in fact, NaHS and GYY caused a mild inhibition of whole-cell Ca 2+ currents. Rather, the facilitation of exocytosis seemed to be associated to an augmented basal [Ca 2+ ] c and the K + -elicited [Ca 2+ ] c transients; such effects of H 2 S donors are aborted by cyclopiazonic acid (CPA), that causes endoplasmic reticulum (ER) Ca 2+ depletion through sarcoendoplasmic reticulum Ca2+ ATPase inhibition and by protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that impedes the ability of mitochondria to sequester cytosolic Ca 2+ during cell depolarization. Inasmuch as CPA and FCCP reversed the facilitation of secretion triggered by K + in the presence of NaHS and GYY, is seems that such facilitation is tightly coupled to Ca 2+ handling by the ER and mitochondria. On the basis of these results, we propose that H 2 S regulates catecholamine secretory responses triggered by K + in BCCs by (i) mobilisation of ER Ca 2+ and (ii) interference with mitochondrial Ca 2+ circulation. In so doing, the clearance of the [Ca 2+ ] c transient will be delayed and the Ca 2+ -dependent trafficking of secretory vesicles will be enhanced to overfill the secretory machinery with new vesicles to enhance exocytosis.

Growth inhibition and differentiation of murine melanoma B16-BL6 cells caused by the combination of cisplatin and caffeine.

PubMed

Tsuchiya, H; Tomita, K; Yasutake, H; Ueda, Y; Tanaka, M; Sasaki, T

1989-12-01

We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16-BL6 cells in vitro. When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G2/M phase, with a partial G2 block. The addition of 2.0 mM caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16-BL6 cells treated with cisplatin to differentiate, and this inhibited growth.

Growth Inhibition and Differentiation of Murine Melanoma B16‐BL6 Cells Caused by the Combination of Cisplatin and Caffeine

PubMed Central

Tomita, Katsuro; Yasutake, Hidetoshi; Ueda, Yoshimichi; Tanaka, Motohiro; Sasaki, Takuma

1989-01-01

We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16‐BL6 cells in vitro. When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G2/M phase, with a partial G2 block. The addition of 2.0 mM caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16‐BL6 cells treated with cisplatin to differentiate, and this inhibited growth. PMID:2516852

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, Joshua R.; Marty, M. Sue; Atchison, William D.

2005-11-01

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 {mu}M MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 {mu}M MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in amore » proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca{sup 2+} and non-Ca{sup 2+} divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 {mu}M MeHg, 97.7% of Purkinje cells were viable. At 3 {mu}M MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 {mu}M MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca{sup 2+} divalent cation released by MeHg in Purkinje neurons.« less

Lymphocyte functional antigens stabilize agglutination between Reed-Sternberg cells and T cells, but are not responsible for homotypic binding of Hodgkin's Reed-Sternberg cells.

PubMed Central

Hsu, S. M.; Hsu, P. L.

1990-01-01

The neoplastic (Hodgkin's Reed-Sternberg [H-RS]) cells in Hodgkin's disease (HD) are known for their unique capacity to form rosettes with unprimed T cells. Recently, a family of leukocyte-adherence molecules (LFA-1 and LFA-2) has been identified on T lymphocytes. The molecules bind to intercellular-adhesion molecules (ICAMs) and to LFA-3, respectively, which are associated with antigen-presenting cells. In this study, the authors examined whether these molecules are responsible for the homotypic and heterotypic agglutination that occurs in the cultured H-RS cells HDLM-1, HDLM-1d, and KM-H2. Despite their similar expressions of LFA-3 and ICAM-1, the different H-RS cells tested showed different growth patterns in culture. HDLM-1 cells grew singly, whereas HDLM-1d and KM-H2 cells grew in clumps. The HDLM-1 cells formed clumps when mixed with peripheral-blood T lymphocytes, cells of two lymphoblastic T-cell lines (MOLT-3 and MOLT-4), and cells of two monocyte lines (ML-1 and U-937). The addition of anti-LFA and ICAM-1 antibodies to cultures did not result in disassembly of the homotypic clusters of HDLM-1d or KM-H2 cells and it did not cause any significant changes in the size of heterotypic clusters or in the timing of cluster formation of HDLM-1 cells with other types of cells. In all studies, the cell clusters formed during homotypic and heterotypic aggregation were disassembled only minimally by cell shearing with pipetting. The disaggregation by pipetting was slightly more prominent in the presence of antibodies than was that of control cultures. However, in no case did the use of monoclonal antibodies (MAbs) and cell shearing cause complete disaggregation of homotypic and heterotypic clusters. The result seems to suggest that binding between H-RS cells and T cells and between H-RS cells and monocytes is not mediated primarily by LFAs and ICAMs, but that the binding may be strengthened in the presence of these molecules. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1698024

Pirfenidone inhibits p38-mediated generation of procoagulant microparticles by human alveolar epithelial cells.

PubMed

Neri, Tommaso; Lombardi, Stefania; Faìta, Francesca; Petrini, Silvia; Balìa, Cristina; Scalise, Valentina; Pedrinelli, Roberto; Paggiaro, Pierluigi; Celi, Alessandro

2016-08-01

Pirfenidone is a drug recently approved for idiopathic pulmonary fibrosis but its mechanisms of action are partially unknown. We have previously demonstrated that the airways of patients with idiopathic pulmonary fibrosis contain procoagulant microparticles that activate coagulation factor X to its active form, Xa, a proteinase that signals fibroblast growth and differentiation, thus potentially contributing to the pathogenesis of the disease. We also reported that in vitro exposure of human alveolar cells to H2O2 causes microparticle generation. Since p38 activation is involved in microparticle generation in some cell models and p38 inhibition is one of the mechanisms of action of pirfenidone, we investigated the hypothesis that H2O2-induced generation of microparticles by alveolar cells is dependent on p38 phosphorylation and is inhibited by pirfenidone. H2O2 stimulation of alveolar cells caused p38 phosphorylation that was inhibited by pirfenidone. The drug also inhibited H2O2 induced microparticle generation as assessed by two independent methods (solid phase thrombin generation and flow cytometry). The shedding of microparticle-bound tissue factor activity was also inhibited by pirfenidone. Inhibition of p38-mediated generation of procoagulant microparticle is a previously unrecognized mechanism of action of the antifibrotic drug, pirfenidone. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woo, Seon Rang; Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705; Park, Jeong-Eun

2012-01-06

Highlights: Black-Right-Pointing-Pointer Under conditions of telomere erosion, cells become extremely sensitive to H{sub 2}O{sub 2}. Black-Right-Pointing-Pointer Chromosomal regions adjacent to telomeres are cleaved by H{sub 2}O{sub 2} under such conditions. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} thus causes multichromosomal fusions and generation of small chromosomal fragments. Black-Right-Pointing-Pointer N-acetylcysteine prevents H{sub 2}O{sub 2}-induced chromosomal aberrations. -- Abstract: During genotoxic stress, reactive oxygen species hydrogen peroxide (H{sub 2}O{sub 2}) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H{submore » 2}O{sub 2}, via generation of multichromosomal fusion and chromosomal fragments. H{sub 2}O{sub 2} caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H{sub 2}O{sub 2} cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H{sub 2}O{sub 2}. Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.« less

Cytotoxic Effects of Dimorfolido-N-Trichloroacetylphosphorylamide and Dimorfolido-N-Benzoylphosphorylamide in Combination with C60 Fullerene on Leukemic Cells and Docking Study of Their Interaction with DNA.

PubMed

Prylutska, S; Grynyuk, I; Grebinyk, A; Hurmach, V; Shatrava, Iu; Sliva, T; Amirkhanov, V; Prylutskyy, Yu; Matyshevska, O; Slobodyanik, M; Frohme, M; Ritter, U

2017-12-01

Dimorfolido-N-trichloroacetylphosphorylamide (HL1) and dimorfolido-N-benzoylphosphorylamide (HL2) as representatives of carbacylamidophosphates were synthesized and identified by the methods of IR, 1 H, and 31 P NMR spectroscopy. In vitro HL1 and HL2 at 1 mM concentration caused cell specific and time-dependent decrease of leukemic cell viability. Compounds caused the similar gradual decrease of Jurkat cells viability at 72 h (by 35%). HL1 had earlier and more profound toxic effect as compared to HL2 regardless on leukemic cell line. Viability of Molt-16 and CCRF-CEM cells under the action of HL1 was decreased at 24 h (by 32 and 45%, respectively) with no substantial further reducing up to 72 h. Toxic effect of HL2 was detected only at 72 h of incubation of Jurkat and Molt-16 cells (cell viability was decreased by 40 and 45%, respectively).It was shown that C 60 fullerene enhanced the toxic effect of HL2 on leukemic cells. Viability of Jurkat and CCRF-CEM cells at combined action of C 60 fullerene and HL2 was decreased at 72 h (by 20 and 24%, respectively) in comparison with the effect of HL2 taken separately.In silico study showed that HL1 and HL2 can interact with DNA and form complexes with DNA both separately and in combination with C 60 fullerene. More stable complexes are formed when DNA interacts with HL1 or C 60  + HL2 structure. Strong stacking interactions can be formed between HL2 and C 60 fullerene. Differences in the types of identified bonds and ways of binding can determine distinction in cytotoxic effects of studied compounds.

Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

PubMed Central

Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

2016-01-01

In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

Evolutionary genomics and HIV restriction factors.

PubMed

Pyndiah, Nitisha; Telenti, Amalio; Rausell, Antonio

2015-03-01

To provide updated insights into innate antiviral immunity and highlight prototypical evolutionary features of well characterized HIV restriction factors. Recently, a new HIV restriction factor, Myxovirus resistance 2, has been discovered and the region/residue responsible for its activity identified using an evolutionary approach. Furthermore, IFI16, an innate immunity protein known to sense several viruses, has been shown to contribute to the defense to HIV-1 by causing cell death upon sensing HIV-1 DNA. Restriction factors against HIV show characteristic signatures of positive selection. Different patterns of accelerated sequence evolution can distinguish antiviral strategies--offense or defence--as well as the level of specificity of the antiviral properties. Sequence analysis of primate orthologs of restriction factors serves to localize functional domains and sites responsible for antiviral action. We use recent discoveries to illustrate how evolutionary genomic analyses help identify new antiviral genes and their mechanisms of action.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

PubMed

Arpornmaeklong, Premjit; Pressler, Michael J

2018-01-01

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis

DOE PAGES

Wang, Wei; Li, Eryang; Porth, Ilga; ...

2016-02-02

Among the R2R3 MYB transcription factors that involve in the regulation of secondary cell wall formation in Arabidopsis, MYB46 alone is sufficient to induce the entire secondary cell wall biosynthesis program. PtrMYB021, the poplar homolog of MYB46, has been reported to regulate secondary cell wall formation when expressed in Arabidopsis. We report here that spatially and temporally restricted expression of PtrMYB021 is critical for its function in regulating secondary cell wall formation. By using quantitative RT-PCR, we found that PtrMYB021 was expressed primarily in xylem tissues. When expressed in Arabidopsis under the control of PtrCesA8, but not the 35S promoter,more » PtrMYB021 increased secondary cell wall thickness, which is likely caused by increased lignification as well as changes in cell wall carbohydrate composition. Consistent with this, elevated expression of lignin and cellulose biosynthetic genes were observed in the transgenic plants. Finally, when expressed in Arabidopsis protoplasts as fusion proteins to the Gal4 DNA binding domain, PtrMYB021 activated the reporter gene Gal4-GUS. In summary, our results suggest that PtrMYB021 is a transcriptional activator, and spatially and temporally restricted expression of PtrMYB021 in Arabidopsis regulates secondary cell wall formation by activating a subset of secondary cell wall biosynthesis genes.« less

Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Li, Eryang; Porth, Ilga

Among the R2R3 MYB transcription factors that involve in the regulation of secondary cell wall formation in Arabidopsis, MYB46 alone is sufficient to induce the entire secondary cell wall biosynthesis program. PtrMYB021, the poplar homolog of MYB46, has been reported to regulate secondary cell wall formation when expressed in Arabidopsis. We report here that spatially and temporally restricted expression of PtrMYB021 is critical for its function in regulating secondary cell wall formation. By using quantitative RT-PCR, we found that PtrMYB021 was expressed primarily in xylem tissues. When expressed in Arabidopsis under the control of PtrCesA8, but not the 35S promoter,more » PtrMYB021 increased secondary cell wall thickness, which is likely caused by increased lignification as well as changes in cell wall carbohydrate composition. Consistent with this, elevated expression of lignin and cellulose biosynthetic genes were observed in the transgenic plants. Finally, when expressed in Arabidopsis protoplasts as fusion proteins to the Gal4 DNA binding domain, PtrMYB021 activated the reporter gene Gal4-GUS. In summary, our results suggest that PtrMYB021 is a transcriptional activator, and spatially and temporally restricted expression of PtrMYB021 in Arabidopsis regulates secondary cell wall formation by activating a subset of secondary cell wall biosynthesis genes.« less

T cell receptor cross-reactivity between similar foreign and self peptides influences naïve cell population size and autoimmunity

PubMed Central

Nelson, Ryan W.; Beisang, Daniel; Tubo, Noah J.; Dileepan, Thamotharampillai; Wiesner, Darin L.; Nielsen, Kirsten; Wüthrich, Marcel; Klein, Bruce S.; Kotov, Dmitri I.; Spanier, Justin A.; Fife, Brian T.; Moon, James J.; Jenkins, Marc K.

2014-01-01

SUMMARY T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naïve CD4+ T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant since systemic expression of a self peptide reduced the size of a naïve cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naïve T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations, and may allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection. PMID:25601203

Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells

PubMed Central

Kumar, Nathan; Richter, Jenna; Cutts, Josh; Bush, Kevin T; Trujillo, Cleber; Nigam, Sanjay K; Gaasterland, Terry; Brafman, David; Willert, Karl

2015-01-01

The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate. DOI: http://dx.doi.org/10.7554/eLife.08413.001 PMID:26554899

An outbreak of food poisoning due to egg yolk reaction-negative Staphylococcus aureus.

PubMed

Miwa, N; Kawamura, A; Masuda, T; Akiyama, M

2001-03-20

An outbreak of staphylococcal food poisoning due to an egg yolk (EY) reaction-negative strain occurred in Japan. Twenty-one of 53 dam construction workers who ate boxed lunches prepared at their company cafeteria became ill, and eight required hospital treatment. The outbreak showed a typical incubation time (1.5-4 h with a median time of 2.7 h) and symptoms (vomiting and diarrhea) of staphylococcal food poisoning. Staphylococcus aureus, which produces staphylococcal enterotoxin (SE) A, was isolated from four fecal specimens of eight patients tested. Scrambled egg in the boxed lunches contained 20-40 ng/g of SEA, and 3.0 x 10(9)/g of viable S. aureus cells that produced this toxin. All isolates from patients and the food were EY reaction-negative, coagulase type II, and showed the same restriction fragment length polymorphism (RFLP) pattern. We concluded that the outbreak was caused by scrambled egg contaminated with EY reaction-negative S. aureus. In Japan, outbreaks of staphylococcal food poisoning are mainly caused by EY reaction-positive S. aureus, and EY reaction-negative colonies grown on agar plates containing EY are usually not analyzed further for detection of S. aureus. The present outbreak suggested that EY reaction-negative isolates should be subjected to further analysis to detect the causative agents of staphylococcal food poisoning.

Human Umbilical Cord Wharton's Jelly Stem Cell Conditioned Medium Induces Tumoricidal Effects on Lymphoma Cells Through Hydrogen Peroxide Mediation.

PubMed

Lin, Hao Daniel; Fong, Chui-Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

2016-09-01

Several groups have reported that human umbilical cord Wharton's jelly stem cells (hWJSCs) possess unique tumoricidal properties against many cancers. However, the exact mechanisms as to how hWJSCs inhibit tumor growth are not known. Recent evidence suggests that exposure of cancer cells to high hydrogen peroxide (H2 O2 ) levels from H2 O2 -releasing drugs causes their death. We therefore explored whether the tumoricidal effect of hWJSCs on lymphoma cells was mediated via H2 O2 . We first exposed lymphoma cells to six different molecular weight cut-off (MWCO) concentrates of hWJSC-conditioned medium (hWJSC-CM) (3, 5, 10, 30, 50, 100 kDa) for 48 h. Since, the 3 kDa-MWCO concentrate showed the greatest cell inhibition we then investigated whether the tumoricidal effect of the specific 3 kDa-MWCO concentrate on two different lymphoma cell lines (Ramos and Toledo) was mediated via accumulation of H2 O2 . We used a battery of assays (MTT, propidium iodide, mitochondria membrane potential, apoptosis, cell cycle, oxidative stress enzymes, hydrogen peroxide, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation) to test this mechanism. The hWJSC-CM-3 kDa MWCO concentrate significantly decreased cell viability and mitochondrial membrane potential and increased cell death and apoptosis in both lymphoma cell lines. There were significant increases in superoxide dismutase with concomitant decreases in glutathione peroxidase, catalase, and thioredoxin peroxidase activities. H2 O2 levels, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation were also significantly increased in both lymphoma cell lines. The results suggested that the hWJSC-CM-3 kDa MWCO concentrate regulates cellular H2 O2 leading to a tumoricidal effect and may thus be a promising anti-lymphoma agent. J. Cell. Biochem. 117: 2045-2055, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Chemopreventive effect of chalcone derivative, L2H17, in colon cancer development.

PubMed

Xu, Shanmei; Chen, Minxiao; Chen, Wenbo; Hui, Junguo; Ji, Jiansong; Hu, Shuping; Zhou, Jianmin; Wang, Yi; Liang, Guang

2015-11-09

Colon cancer is the third most commonly diagnosed cancer and the second leading cause of cancer mortality worldwide. Chalcone and its derivatives are reported to exhibit anti-cancer effects in several cancer cell lines, including colon cancer cells. In addition, chalcones have advantages such as poor interaction with DNA and low risk of mutagenesity. In our previous study, a group of chalcone derivatives were synthesized and exhibited strong anti-inflammatory activities. In this study, we evaluated the anti-cancer effects of the chalcone derivative, L2H17, in colon cancer cells. The cytotoxicities of L2H17 on various colon cancer cell lines were investigated by MTT and clonogenic assay. Cell cycle and apoptosis analysis were performed to evaluate the molecular mechanism of L2H17-mediated inhibition of tumor growth. Also, scratch wound and matrigel invasion experiments were performed to estimate the cell migration and invasion after L2H17 treatment. Finally, we observed the anti-colon cancer effects of L2H17 in vivo. Our data show that compound L2H17 exhibited selective cytotoxic effect on colon cancer cells, via inducing G0/G1 cell cycle arrest and apoptosis in CT26.WT cells. Furthermore, L2H17 treatment decreased cell migration and invasion of CT26.WT cells. In addition, L2H17 possessed marked anti-tumor activity in vivo. The molecular mechanism of L2H17-mediated inhibition of tumor promotion and progression were function through inactivated NF-κB and Akt signaling pathways. All these findings show that L2H17 might be a potential growth inhibitory chalcones derivative for colon cancer cells.

Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses.

PubMed

Jacobs, Sandra A; Pinxteren, Jef; Roobrouck, Valerie D; Luyckx, Ariane; van't Hof, Wouter; Deans, Robert; Verfaillie, Catherine M; Waer, Mark; Billiau, An D; Van Gool, Stefaan W

2013-01-01

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn's disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

Tetraploidization or autophagy: The ultimate fate of senescent human endometrial stem cells under ATM or p53 inhibition.

PubMed

Borodkina, Aleksandra V; Shatrova, Alla N; Deryabin, Pavel I; Grukova, Anastasiya A; Nikolsky, Nikolay N; Burova, Elena B

2016-01-01

Previously we demonstrated that endometrium-derived human mesenchymal stem cells (hMESCs) via activation of the ATM/p53/p21/Rb pathway enter the premature senescence in response to oxidative stress. Down regulation effects of the key components of this signaling pathway, particularly ATM and p53, on a fate of stressed hMESCs have not yet been investigated. In the present study by using the specific inhibitors Ku55933 and Pifithrin-α, we confirmed implication of both ATM and p53 in H(2)O(2)-induced senescence of hMESCs. ATM or p53 down regulation was shown to modulate differently the cellular fate of H(2)O(2)-treated hMESCs. ATM inhibition allowed H(2)O(2)-stimulated hMESCs to escape the permanent cell cycle arrest due to loss of the functional ATM/p53/p21/Rb pathway, and induced bypass of mitosis and re-entry into S phase, resulting in tetraploid cells. On the contrary, suppression of the p53 transcriptional activity caused a pronounced cell death of H(2)O(2)-treated hMESCs via autophagy induction. The obtained data clearly demonstrate that down regulation of ATM or p53 shifts senescence of human endometrial stem cells toward tetraploidization or autophagy.

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress.

PubMed

Bhaskaran, Natarajan; Shukla, Sanjeev; Kanwal, Rajnee; Srivastava, Janmejai K; Gupta, Sanjay

2012-06-27

Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. The cytoprotective effect of chamomile was examined on H(2)O(2)-induced cellular stress in RAW 264.7 murine macrophages. RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H(2)O(2). Treatment with 50μM H(2)O(2) for 6h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20μg/mL for 16h followed by H(2)O(2) treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties. Copyright © 2012 Elsevier Inc. All rights reserved.

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

PubMed Central

Bhaskaran, Natarajan; Shukla, Sanjeev; Kanwal, Rajnee; Srivastava, Janmejai K; Gupta, Sanjay

2012-01-01

Aims Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. Main Methods The cytoprotective effect of chamomile was examined on H2O2-induced cellular stress in RAW 264.7 murine macrophages. Key Findings RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H2O2. Treatment with 50 μM H2O2 for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20 μg/mL for 16 h followed by H2O2 treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. Significance These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties. PMID:22683429

Pregnancy interceptive efficacy and biological profile of 3-amino-6,7-dimethoxy-1H-pyrazolo [3,4-b] quinoline (compound 85/83) in rodents.

PubMed

Mehrotra, P K; Shukla, R; Dwivedi, A; Srivastava, R P; Bhat, N; Seth, M; Bhaduri, A P; Kamboj, V P

1991-05-01

Administration of compound 85/83 during the peri- and post-implantation period intercepted pregnancy in hamster and guinea pig by parenteral route and in hamster by oral route also. The m.e.d. for hamster and guinea pig was 10 and 20 mg/kg, respectively; lower doses were less effective. Restricting the administration to early post-implantation schedule interrupted pregnancy partially in both species. The compound was, however, ineffective in rat and in the pre-implantation schedule (days 1-4 post-coitum) in hamster. When tested in vitro on growing trophoblasts at 13.8 x 10(-5) M concentration, it prevented growth and caused degeneration of the cells within 24 h; lower concentration (9.2 x 10(-5) M) was less effective. The compound was found to be devoid of estrogenic, antiestrogenic, progestational and antiprogestational properties in conventional bioassays. In hormone competition assays, its relative binding affinity (RBA) to estrogen receptor was negligible (0.002% of estradiol-17 beta), while for uterine cytosol progesterone receptors in rabbit and hamster was 0.06 and 0.08% of progesterone, respectively. The compound 85/83 appears to intercept pregnancy by interfering with development of trophoblast cells.

Mitochondrial dysfunction in H9c2 cells during ischemia and amelioration with Tribulus terrestris L.

PubMed

Reshma, P L; Sainu, Neethu S; Mathew, Anil K; Raghu, K G

2016-05-01

The present study investigates the protective effect of partially characterized Tribulus terrestris L. fruit methanol extract against mitochondrial dysfunction in cell based (H9c2) myocardial ischemia model. To induce ischemia, the cells were maintained in an ischemic buffer (composition in mM -137 NaCl, 12 KCl, 0.5 MgCl2, 0.9 CaCl2, 20 HEPES, 20 2-deoxy-d-glucose, pH-6.2) at 37°C with 0.1% O2, 5% CO2, and 95% N2 in a hypoxia incubator for 1h. Cells were pretreated with various concentrations of T. terrestris L. fruit methanol extract (10 and 25μg/ml) and Cyclosporin A (1μM) for 24h prior to the induction of ischemia. Different parameters like lactate dehydrogenase release, total antioxidant capacity, glutathione content and antioxidant enzymes were investigated. Studies were conducted on mitochondria by analyzing alterations in mitochondrial membrane potential, integrity, and dynamics (fission and fusion proteins - Mfn1, Mfn2, OPA1, Drp1 and Fis1). Various biochemical processes in mitochondria like activity of electron transport chain (ETC) complexes, oxygen consumption and ATP production was measured. Ischemia for 1h caused a significant (p≤0.05) increase in LDH leakage, decrease in antioxidant activity and caused mitochondrial dysfunction. T. terrestris L. fruit methanol extract pretreatment was found effective in safeguarding mitochondria via its antioxidant potential, mediated through various bioactives. HPLC of T. terrestris L. fruit methanol extract revealed the presence of ferulic acid, phloridzin and diosgenin. T. terrestris L. fruit ameliorate ischemic insult in H9c2 cells by safeguarding mitochondrial function. This validates the use of T. terrestris L. against heart disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Intestinal morphology adjustments caused by dietary restriction improves the nutritional status during the aging process of rats.

PubMed

de Oliveira Belém, Mônica; Cirilo, Carla Possani; de Santi-Rampazzo, Ana Paula; Schoffen, João Paulo Ferreira; Comar, Jurandir Fernando; Natali, Maria Raquel Marçal; de Almeida Araújo, Eduardo José

2015-09-01

During the aging process, the body's systems change structurally and loss of function can occur. Ingesting a smaller amount of food has been considered a plausible proposal for increased longevity with the quality of life. However, the effects of dietary restriction (DR) during aging are still poorly understood, especially for organs of the digestive system. This study aimed to describe the body weight, oxidative status and possible morphological changes of the intestinal wall of rats submitted to DR during the aging process (7 to 18months old). Twelve 7-month-old male Wistar rats fed ad libitum since birth were assigned to two groups: control group (CG, n=6) fed ad libitum from 7 to 18months old; and dietary restriction group (DRG, n=6) fed 50% of the amount of chow consumed by the CG from 7 to 18months old. The body weight, feed and water intake were monitored throughout the experiment. Blood, periepididymal adipose tissue (PAT) and retroperitoneal adipose tissue (RAT), and the small intestine were collected at 18months old. The blood was collected to evaluate its components and oxidative status. Sections from the duodenum and ileum were stained with HE, PAS and AB pH2.5 for morphometric analyses of the intestinal wall components, and to count intraepithelial lymphocytes (IELs), goblet cells and cells in mitosis in the epithelium. DR rats showed a reduction in weight, naso-anal length, PAT, RAT and intestinal length; however, they consumed more water. Blood parameters indicate that the DR rats remained well nourished. In addition, they showed lower lipid peroxidation. Hypertrophy of the duodenal mucosa and atrophy of the ileal mucosa were observed. The number of goblet cells and IELs was reduced, but the mitotic index remained unaltered in both duodenum and ileum. In conclusion, 50% dietary restriction for rats from 7 to 18months old contributed to improving their nutritional parameters but, to achieve this, adjustments were required in the structure of the body weight and morphology of the small intestine. Copyright © 2015 Elsevier Inc. All rights reserved.

Abnormal pulmonary function in adults with sickle cell anemia.

PubMed

Klings, Elizabeth S; Wyszynski, Diego F; Nolan, Vikki G; Steinberg, Martin H

2006-06-01

Pulmonary complications of sickle cell anemia (Hb-SS) commonly cause morbidity, yet few large studies of pulmonary function tests (PFTs) in this population have been reported. PFTs (spirometry, lung volumes, and diffusion capacity for carbon monoxide [DLCO]) from 310 adults with Hb-SS were analyzed to determine the pattern of pulmonary dysfunction and their association with other systemic complications of sickle cell disease. Raw PFT data were compared with predicted values. Each subject was subclassified into one of five groups: obstructive physiology, restrictive physiology, mixed obstructive/restrictive physiology, isolated low DLCO, or normal. The association between laboratory data of patients with decreased DLCO or restrictive physiology and those of normal subjects was assessed by multivariate linear regression. Normal PFTs were present in only 31 of 310 (10%) patients. Overall, adults with Hb-SS were characterized by decreased total lung capacities (70.2 +/- 14.7% predicted) and DLCO (64.5 +/- 19.9%). The most common PFT patterns were restrictive physiology (74%) and isolated low DLCO (13%). Decreased DLCO was associated with thrombocytosis (p = 0.05), with hepatic dysfunction (elevated alanine aminotransferase; p = 0.07), and a trend toward renal dysfunction (elevated blood urea nitrogen and creatinine; p = 0.05 and 0.07, respectively). Pulmonary function is abnormal in 90% of adult patients with Hb-SS. Common abnormalities include restrictive physiology and decreased DLCO. Decreased DLCO may indicate more severe sickle vasculopathy characterized by impaired hepatic and renal function.

Ginger Oleoresin Alleviated γ-Ray Irradiation-Induced Reactive Oxygen Species via the Nrf2 Protective Response in Human Mesenchymal Stem Cells

PubMed Central

Ji, Kaihua; Li, Qing; Shi, Yang; Xu, Chang; Wang, Yan; Du, Liqing

2017-01-01

Unplanned exposure to radiation can cause side effects on high-risk individuals; meanwhile, radiotherapies can also cause injury on normal cells and tissues surrounding the tumor. Besides the direct radiation damage, most of the ionizing radiation- (IR-) induced injuries were caused by generation of reactive oxygen species (ROS). Human mesenchymal stem cells (hMSCs), which possess self-renew and multilineage differentiation capabilities, are a critical population of cells to participate in the regeneration of IR-damaged tissues. Therefore, it is imperative to search effective radioprotectors for hMSCs. This study was to demonstrate whether natural source ginger oleoresin would mitigate IR-induced injuries in human mesenchymal stem cells (hMSCs). We demonstrated that ginger oleoresin could significantly reduce IR-induced cytotoxicity, ROS generation, and DNA strand breaks. In addition, the ROS-scavenging mechanism of ginger oleoresin was also investigated. The results showed that ginger oleoresin could induce the translocation of Nrf2 to cell nucleus and activate the expression of cytoprotective genes encoding for HO-1 and NQO-1. It suggests that ginger oleoresin has a potential role of being an effective antioxidant and radioprotective agent. PMID:29181121

Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C.

PubMed

Peng, Chaohua; Zhu, Gaochun; Liu, Xiangqian; Li, He

2018-04-30

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

Fasting cycles potentiate the efficacy of gemcitabine treatment in in vitro and in vivo pancreatic cancer models

PubMed Central

Mazza, Tommaso; Panebianco, Concetta; Saracino, Chiara; Pereira, Stephen P.; Graziano, Paolo; Pazienza, Valerio

2015-01-01

Background/aims Pancreatic cancer (PC) is ranked as the fourth leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, a modest impact on the outcome of the disease is observed so far. Short-term fasting cycles have been shown to potentiate the efficacy of chemotherapy against glioma. The aim of this study was to assess the effect of fasting cycles on the efficacy of gemcitabine, a standard treatment for PC patients, in vitro and in an in vivo pancreatic cancer mouse xenograft model. Materials and Methods BxPC-3, MiaPaca-2 and Panc-1 cells were cultured in standard and fasting mimicking culturing condition to evaluate the effects of gemcitabine. Pancreatic cancer xenograft mice were subjected to 24h starvation prior to gemcitabine injection to assess the tumor volume and weight as compared to mice fed ad libitum. Results Fasted pancreatic cancer cells showed increased levels of equilibrative nucleoside transporter (hENT1), the transporter of gemcitabine across the cell membrane, and decreased ribonucleotide reductase M1 (RRM1) levels as compared to those cultured in standard medium. Gemcitabine was more effective in inducing cell death on fasted cells as compared to controls. Consistently, xenograft pancreatic cancer mice subjected to fasting cycles prior to gemcitabine injection displayed a decrease of more than 40% in tumor growth. Conclusion Fasting cycles enhance gemcitabine effect in vitro and in the in vivo PC xenograft mouse model. These results suggest that restrictive dietary interventions could enhance the efficacy of existing cancer treatments in pancreatic cancer patients. PMID:26176887

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guseva, Daria; Hannover Medical School, Hannover; Rizvanov, Albert A.

2014-09-05

Highlights: • Gene and cell-based therapies comprise innovative aspects of regenerative medicine. • Genetically modified hUCB-MCs enhanced differentiation of cells in a mouse model of ALS. • Stem cells successfully transformed into micro-glial and endothelial lines in spinal cords. • Over-expressing oct4 and sox2 also induced production of neural marker PGP9.5. • Formation of new nerve cells, secreting trophic factors and neo-vascularisation could improve symptoms in ALS. - Abstract: Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignantmore » transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.« less

Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury

PubMed Central

Lazrak, Ahmed; Creighton, Judy; Yu, Zhihong; Komarova, Svetlana; Doran, Stephen F.; Aggarwal, Saurabh; Emala, Charles W.; Stober, Vandy P.; Trempus, Carol S.; Garantziotis, Stavros

2015-01-01

Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca2+, and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca2+, blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca2+ channels of airway smooth muscle cells, increasing their contractility and thus causing AHR. PMID:25747964

Preparation of a Two-Photon Fluorescent Probe for Imaging H2O2 in Lysosomes in Living Cells and Tissues.

PubMed

Ren, Mingguang; Deng, Beibei; Kong, Xiuqi; Tang, Yonghe; Lin, Weiying

2017-01-01

Hydrogen peroxide (H 2 O 2 ) plays important roles in many physiological and pathological processes. At the cellular organelle level, the abnormal concentrations of H 2 O 2 in the lysosomes may cause redox imbalance and the loss of the critical functions of the lysosomes. Herein, we describe the preparation of a potent lysosome-targeted two-photon fluorescent probe (Lyso-HP) for the detection of H 2 O 2 in the lysosomes in the living cells. This unique fluorescent probe can also be employed to effectively detect H 2 O 2 in the living tissues using two-photon fluorescence microscopy.

Peroxiredoxins are important for the regulation of hydrogen peroxide concentrations in ticks and tick cell line.

PubMed

Kusakisako, Kodai; Hernandez, Emmanuel Pacia; Talactac, Melbourne Rio; Yoshii, Kentaro; Umemiya-Shirafuji, Rika; Fujisaki, Kozo; Tanaka, Tetsuya

2018-03-17

Ticks are obligate hematophagous ectoparasites, as they need to feed blood from vertebrate hosts for development. Host blood contains high levels of iron. Host-derived iron may lead to high levels of reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ). Since a high concentration of H 2 O 2 causes serious damage to organisms, this molecule is known to be a harmful chemical compound for aerobic organisms. On the other hand, the transparent method is compatible with chemical fluorescent probes. Therefore, we tried to establish the visualizing method for H 2 O 2 in unfed tick tissues. The combination method of a chemical fluorescent probe (BES-H 2 O 2 -Ac) with the transparent method, Scale, demonstrated in unfed tick tissues that H 2 O 2 and paraquat could induce oxidative stress in the tissues, such as the midgut and ovary. In addition, an H 2 O 2 detection method using BES-H 2 O 2 -Ac was established in Ixodes scapularis embryo-derived cell line (ISE6) in vitro to evaluate the antioxidant activity of peroxiredoxins (PRXs), H 2 O 2 scavenging enzymes, against H 2 O 2 in the cells. The effects of paraquat in ISE6 cells were also observed in the PRXs gene-silenced ISE6 cells. A high intensity of H 2 O 2 fluorescence induced by paraquat was observed in the PRX gene-knockdowned cells. These results suggest that H 2 O 2 and paraquat act as an H 2 O 2 inducer, and PRX genes are important for the regulation of the H 2 O 2 concentration in unfed ticks and ISE6 cells. Therefore, this study contributes to the search for H 2 O 2 visualization in ticks and tick cell line and furthers understanding of the tick's oxidative stress induced by H 2 O 2 . Copyright © 2018 Elsevier GmbH. All rights reserved.

Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2).

PubMed

Pooja, Sharma; Pushpanathan, Muthuirulan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2015-01-01

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction.

Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2)

PubMed Central

Pooja, Sharma; Pushpanathan, Muthuirulan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2015-01-01

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction. PMID:26431539

Free-flow zone electrophoresis: a novel approach and scale-up for preparative protein separation.

PubMed

Poggel, M; Melin, T

2001-04-01

Different continuously working free-flow zone electrophoresis (FFZE) chambers have already been developed [1, 2]. All of them deal with the problem of distinctive Joule heating. The resulting temperature gradients cause an unstable density field which leads to thermal convection and thus to an intermixing of the different fractions within the chamber. The most promising and simple approach to stabilize the flow is to build chambers with one very small dimension (e.g., h = 0.5 mm) to assure efficient heat withdrawal. This in turn presents substantial disadvantages, namely limited throughput and restricted scale-up potential. The novel approach combines a simplified design and assembly with the possibility of straightforward scale-up. It still operates with one small dimension (d = 1-2 mm) to handle the Joule heating. Here, however, not the dimension perpendicular to the electric field but the dimension parallel to the electric field (separation distance) is chosen as the smallest dimension. The efficiency of the new device is shown by the separation of bovine serum albumin (BSA) and cytochrome c with an overall protein throughput of up to 1.1 g/h, using a cell with a separation volume of less than 20 mL.

Clozapine protects PC-12 cells from death due to oxidative stress induced by hydrogen peroxide via a cell-type specific mechanism involving inhibition of extracellular signal-regulated kinase phosphorylation.

PubMed

Magliaro, Brian C; Saldanha, Colin J

2009-08-04

Recent evidence suggests that some atypical antipsychotic drugs may protect against oxidative stress and consequent neurodegeneration by mechanisms that remain unclear. Using the neuron-like rat pheochromocytoma (PC-12) cell line, Clozapine and N-desmethylclozapine were tested for their ability to protect against cell death due to oxidative stress induced by hydrogen peroxide (H(2)O(2)). These drugs demonstrated significant protection of PC-12 cells, as measured by both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Alamar Blue cell viability assays. However, neither viability assay detected a protective effect of Clozapine on human embryonic kidney (HEK293), rat primary cortical neurons, or human neuroblastoma (SH-SY5Y) exposed to H(2)O(2) treatment. The mechanism of protection involves a PC-12 cell-specific differential response to H(2)O(2) treatment vs. the other cell lines. Pre-treatment with 250 microM or 125 microM diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, unexpectedly showed protection of the PC-12 cells from H(2)O(2) treatment. Western blots revealed that Clozapine, N-desmethylclozapine, and DETC reduce the phosphorylation of extracellular signal-regulated kinase (ERK) that is caused by H(2)O(2) exposure in PC-12 cells. In both HEK293 and SH-SY5Y cells, H(2)O(2) exposure did not increase ERK phosphorylation over control, demonstrating a different response to H(2)O(2) vs. PC-12 cells, and explaining why Clozapine could not protect these cells. Also, U0126, a specific MEK inhibitor, was able to protect PC-12 cells from H(2)O(2) exposure, showing that inhibiting ERK phosphorylation is sufficient to provide protection. Cumulatively, these results indicate that Clozapine, N-desmethylclozapine, DETC, and U0126 protect PC-12 cells by blocking the cell-type specific H(2)O(2) induced increase in ERK phosphorylation.

The role of the vagus nerve in the generation of cardiorespiratory interactions in a neotropical fish, the pacu, Piaractus mesopotamicus.

PubMed

Leite, Cleo Alcantara Costa; Taylor, E W; Guerra, C D R; Florindo, L H; Belão, T; Rantin, F T

2009-08-01

The role of the vagus nerve in determining heart rate (f(H)) and cardiorespiratory interactions was investigated in a neotropical fish, Piaractus mesopotamicus. During progressive hypoxia f(H) initially increased, establishing a 1:1 ratio with ventilation rate (f(R)). Subsequently there was a hypoxic bradycardia. Injection of atropine abolished a normoxic inhibitory tonus on the heart and the f(H) adjustments during progressive hypoxia, confirming that they are imposed by efferent parasympathetic inputs via the vagus nerve. Efferent activity recorded from the cardiac vagus in lightly anesthetized normoxic fish included occasional bursts of activity related to spontaneous changes in ventilation amplitude, which increased the cardiac interval. Restricting the flow of aerated water irrigating the gills resulted in increased respiratory effort and bursts of respiration-related activity in the cardiac vagus that seemed to cause f(H) to couple with f(R). Cell bodies of cardiac vagal pre-ganglionic neurons were located in two distinct groups within the dorsal vagal motor column having an overlapping distribution with respiratory motor-neurons. A small proportion of cardiac vagal pre-ganglionic neurons (2%) was in scattered positions in the ventrolateral medulla. This division of cardiac vagal pre-ganglionic neurons into distinct motor groups may relate to their functional roles in determining cardiorespiratory interactions.

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

PubMed Central

Throsby, Mark; van den Brink, Edward; Jongeneelen, Mandy; Poon, Leo L. M.; Alard, Philippe; Cornelissen, Lisette; Bakker, Arjen; Cox, Freek; van Deventer, Els; Guan, Yi; Cinatl, Jindrich; ter Meulen, Jan; Lasters, Ignace; Carsetti, Rita; Peiris, Malik; de Kruif, John; Goudsmit, Jaap

2008-01-01

Background The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. Methods and Findings Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM+ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. Conclusions The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM+ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens. PMID:19079604

Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA.

PubMed

Miri-Moghaddam, Ebrahim; Deezagi, Abdolkhaleg; Soheili, Zahra Sohaila; Shariati, Parvin

2010-01-01

The close correlation between telomerase activity and human telomerase reverse transcriptase (hTERT) expression has made hTERT to be considered as a selective molecular target for human cancer therapy. In this study, the ability of short-interfering RNA (siRNA) to downregulate hTERT expression and its correlation with cell growth and apoptosis in the promyelocytic cell line HL-60 was evaluated. hTERT siRNA was designed and transfected to HL-60. hTERT mRNA expression, cell proliferation and apoptotic cells were measured. The results indicated that hTERT siRNA resulted in 97.2 ± 0.6% downregulation of the hTERT mRNA content; inhibition of the cell proliferation rate was about 52.8 ± 2.3% and the apoptotic index of cells was 30.5 ± 1.5%. hTERT plays an essential role in cell proliferation and control of the viability of leukemic cells, thus promising the development of drugs for leukemia. Copyright © 2010 S. Karger AG, Basel.

hMSH5 Facilitates the Repair of Camptothecin-induced Double-strand Breaks through an Interaction with FANCJ*

PubMed Central

Xu, Yang; Wu, Xiling; Her, Chengtao

2015-01-01

Replication stress from stalled or collapsed replication forks is a major challenge to genomic integrity. The anticancer agent camptothecin (CPT) is a DNA topoisomerase I inhibitor that causes fork collapse and double-strand breaks amid DNA replication. Here we report that hMSH5 promotes cell survival in response to CPT-induced DNA damage. Cells deficient in hMSH5 show elevated CPT-induced γ-H2AX and RPA2 foci with concomitant reduction of Rad51 foci, indicative of impaired homologous recombination. In addition, CPT-treated hMSH5-deficient cells exhibit aberrant activation of Chk1 and Chk2 kinases and therefore abnormal cell cycle progression. Furthermore, the hMSH5-FANCJ chromatin recruitment underlies the effects of hMSH5 on homologous recombination and Chk1 activation. Intriguingly, FANCJ depletion desensitizes hMSH5-deficient cells to CPT-elicited cell killing. Collectively, our data point to the existence of a functional interplay between hMSH5 and FANCJ in double-strand break repair induced by replication stress. PMID:26055704

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition.

PubMed

Lee, Chung Soo; Kim, Yun Jeong; Ko, Hyun Hee; Han, Eun Sook

2005-07-15

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H2O2) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H2O2 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H2O2-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H2O2 in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H2O2 and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H2O2 against PC12 cells. Ethanol may enhance the H2O2-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.

Haemophilus ducreyi Partially Activates Human Myeloid Dendritic Cells▿

PubMed Central

Banks, Keith E.; Humphreys, Tricia L.; Li, Wei; Katz, Barry P.; Wilkes, David S.; Spinola, Stanley M.

2007-01-01

Dendritic cells (DC) orchestrate innate and adaptive immune responses to bacteria. How Haemophilus ducreyi, which causes genital ulcers and regional lymphadenitis, interacts with DC is unknown. H. ducreyi evades uptake by polymorphonuclear leukocyte and macrophage-like cell lines by secreting LspA1 and LspA2. Many H. ducreyi strains express cytolethal distending toxin (CDT), and recombinant CDT causes apoptosis of DC in vitro. Here, we examined interactions between DC and H. ducreyi 35000HP, which produces LspA1, LspA2, and CDT. In human volunteers infected with 35000HP, the ratio of myeloid DC to plasmacytoid DC was 2.8:1 in lesions, compared to a ratio of 1:1 in peripheral blood. Using myeloid DC derived from monocytes as surrogates for lesional DC, we found that DC infected with 35000HP remained as viable as uninfected DC for up to 48 h. Gentamicin protection and confocal microscopy assays demonstrated that DC ingested and killed 35000HP, but killing was incomplete at 48 h. The expression of LspA1 and LspA2 did not inhibit the uptake of H. ducreyi, despite inactivating Src kinases. Infection of DC with live 35000HP caused less cell surface marker activation than infection with heat-killed 35000HP and lipopolysaccharide (LPS) and inhibited maturation by LPS. However, infection of DC with live bacteria caused the secretion of significantly higher levels of interleukin-6 and tumor necrosis factor alpha than infection with heat-killed bacteria and LPS. The survival of H. ducreyi in DC may provide a mechanism by which the organism traffics to lymph nodes. Partial activation of DC may abrogate the establishment of a full Th1 response and an environment that promotes phagocytosis. PMID:17923525

Identification of HLA-A2 restricted T-cell epitopes within the conserved region of the immunoglobulin G heavy-chain in patients with multiple myeloma.

PubMed

Belle, Sebastian; Han, Fang; Condomines, Maud; Christensen, Olaf; Witzens-Harig, Mathias; Kasper, Bernd; Kleist, Christian; Terness, Peter; Moos, Marion; Cremer, Friedrich; Hose, Dirk; Ho, Anthony D; Goldschmidt, Hartmut; Klein, Bernard; Hundemer, Michael

2008-07-01

The aim of this study is the identification of HLA-A2 restricted T-cell epitopes in the conserved region of the immunoglobulin-G-heavy-chain (IgGH) that can be used for immunotherapy in multiple myeloma (MM) patients. After the IgGH gene sequence was scanned for HLA-A2 restricted T-cell epitopes with a high binding affinity to the MHC-I-complex, promising nona-peptides were synthesized. Peptide specific CD8+ T-cells were generated from peripheral blood mononuclear cells (PBMC) of healthy donors (HD) and patients with MM using peptide pulsed dendritic cells (DC) in vitro. The activation and cytotoxicity of CD8+ T-cells was analyzed by IFN-alpha ELISpot-assay and 51Chromium release-assay. HLA-A2 restriction was proven by blocking T-cell activation with anti-HLA-A2 antibodies. Two HLA-A2 restricted T-cell epitopes-TLVTVSSAS derived from the IgGH-framework-region 4 (FR4) and LMISRTPEV from the constant region (CR)-induced expansion of specific CD8+ T-cells from PBMC in two of three (TLVTVSSAS) and one of three (LMISRTPEV) HD respectively. Specific T-cells were induced from PBMC in two of six (TLVTVSSAS) and eight of 19 (LMISRTPEV) patients with MM. Specific CD8+ T-cells also lysed peptide-pulsed target cells in 51Chromium release-assay. LMISRTPEV specific CD8+ T-cells from MM patients lysed specifically the HLA-A2+ IgG myeloma cell line XG-6. We identified two HLA-A2 restricted T-cell epitopes-TLVTVSSAS and LMISRTPEV--which can yield an expansion of CD8+ T-cells with the ability to kill peptide-loaded target cells and HLA-A2+ IgG+ myeloma cells. We conclude that TLVTVSSAS and LMISRTPEV could be T-cell epitopes for immunotherapy in MM patients.

Tonsillar CD56brightNKG2A+ NK cells restrict primary Epstein-Barr virus infection in B cells via IFN-γ.

PubMed

Jud, Aurelia; Kotur, Monika; Berger, Christoph; Gysin, Claudine; Nadal, David; Lünemann, Anna

2017-01-24

Natural killer (NK) cells constitute the first line of defense against viruses and cancers cells. Epstein-Barr virus (EBV) was the first human virus to be directly implicated in carcinogenesis, and EBV infection is associated with a broad spectrum of B cell lymphomas. How NK cells restrict EBV-associated oncogenesis is not understood. Here, we investigated the efficacies and mechanisms of distinct NK cell subsets from tonsils, the portal of entry of EBV, in limiting EBV infection in naïve, germinal center-associated and memory B cells. We found that CD56bright and NKG2A expression sufficiently characterizes the potent anti-EBV capacity of tonsillar NK cells. We observed restriction of EBV infection in B cells as early as 18 hours after infection. The restriction was most efficient in naïve B cells and germinal center-associated B cells, the B cell subsets that exhibited highest susceptibility to EBV infection in vitro. IFN-γ release by and partially NKp44 engagement of CD56bright and NKG2A positive NK cells mediated the restriction that eventually inhibited B-cell transformation. Thus, harnessing CD56brightNKG2A+ NK cell function might be promising to improve treatment strategies that target EBV-associated B cell lymphomas.

Pigeon paramyxovirus type 1 from a fatal human case induces pneumonia in experimentally infected cynomolgus macaques (Macaca fascicularis).

PubMed

Kuiken, Thijs; Buijs, Pascal; van Run, Peter; van Amerongen, Geert; Koopmans, Marion; van den Hoogen, Bernadette

2017-11-21

Although avian paramyxovirus type 1 is known to cause mild transient conjunctivitis in human beings, there are two recent reports of fatal respiratory disease in immunocompromised human patients infected with the pigeon lineage of the virus (PPMV-1). In order to evaluate the potential of PPMV-1 to cause respiratory tract disease, we inoculated a PPMV-1 isolate (hPPMV-1/Netherlands/579/2003) from an immunocompromised human patient into three healthy cynomolgus macaques (Macaca fascicularis) and examined them by clinical, virological, and pathological assays. In all three macaques, PPMV-1 replication was restricted to the respiratory tract and caused pulmonary consolidation affecting up to 30% of the lung surface. Both alveolar and bronchiolar epithelial cells expressed viral antigen, which co-localized with areas of diffuse alveolar damage. The results of this study demonstrate that PPMV-1 is a primary respiratory pathogen in cynomolgus macaques, and support the conclusion that PPMV-1 may cause fatal respiratory disease in immunocompromised human patients.

The L1-type cell adhesion molecule neuroglian influences the stability of neural ankyrin in the Drosophila embryo but not its axonal localization.

PubMed

Bouley, M; Tian, M Z; Paisley, K; Shen, Y C; Malhotra, J D; Hortsch, M

2000-06-15

Ankyrins are linker proteins, which connect various membrane proteins, including members of the L1 family of neural cell adhesion molecules, with the submembranous actin-spectrin skeleton. Here we report the cloning and characterization of a second, novel Drosophila ankyrin gene (Dank2) that appears to be the result of a gene duplication event during arthropod evolution. The Drosophila L1-type protein neuroglian interacts with products from both Drosophila ankyrin genes. Whereas the previously described ankyrin gene is ubiquitously expressed during embryogenesis, the expression of Dank2 is restricted to the nervous system in the Drosophila embryo. The absence of neuroglian protein in a neuroglian null mutant line causes decreased levels of Dank2 protein in most neuronal cells. This suggests that neuroglian is important for the stability of Dank2 protein. However, neuroglian is not required for Dank2 axonal localization. In temperature-sensitive neuroglian mutants in which neuroglian protein is mislocated at the restrictive temperature to an intracellular location in the neuronal soma, Dank2 protein can still be detected along embryonic nerve tracts.

Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29.

PubMed

Bellion, Phillip; Olk, Melanie; Will, Frank; Dietrich, Helmut; Baum, Matthias; Eisenbrand, Gerhard; Janzowski, Christine

2009-10-01

Beneficial health effects of diets containing fruits have partly been attributed to polyphenols which display a spectrum of bioactive effects, including antioxidant activity. However, polyphenols can also exert prooxidative effects in vitro. In this study, polyphenol-mediated hydrogen peroxide (H(2)O(2)) formation was determined after incubation of apple juice extracts (AEs) and polyphenols in cell culture media. Effects of extracellular H(2)O(2 )on total glutathione (tGSH; =GSH + GSSG) and cellular reactive oxygen species (ROS) level of HT-29 cells were studied by coincubation +/- catalase (CAT). AEs ( > or =30 microg/mL) significantly generated H(2)O(2) in DMEM, depending on their composition. Similarly, H(2)O(2) was measured for individual apple polyphenols/degradation products (phenolic acids > epicatechin, flavonols > dihydrochalcones). Highest concentrations were generated by compounds bearing the o-catechol moiety. H(2)O(2) formation was found to be pH dependent; addition of CAT caused a complete decomposition of H(2)O(2) whereas superoxide dismutase was less/not effective. At incubation of HT-29 cells with quercetin (1-100 microM), generated H(2)O(2) slightly contributed to antioxidant cell protection by modulation of tGSH- and ROS-level. In conclusion, H(2)O(2) generation in vitro by polyphenols has to be taken into consideration when interpreting results of such cell culture experiments. Unphysiologically high polyphenol concentrations, favoring substantial H(2)O(2 )formation, are not expected to be met in vivo, even under conditions of high end nutritional uptake.

Application of spectroscopy (1HMRS) to assess liver metabolite concentrations in rats with intrauterine growth restriction.

PubMed

Wang, Tao; Chen, Pingyang; Bian, Dujun; Chen, Juncao

2017-04-01

Proton magnetic resonance spectroscopy ( 1 H-MRS) measurement of liver metabolism in intrauterine growth restriction rats has seldom been reported. This study investigated the application of 1 H-MRS in assessing liver metabolism in newborn pups that experienced intrauterine growth restriction. Intra-uterine growth restriction was established by feeding rats low-protein diets during pregnancy. Newborn pups received conventional magnetic resonance imaging and 1 H-MRS using a 3.0T whole body MR scanner at 3, 8 and 12 weeks post birth. The success rate of 1 H-MRS was 83.33%. Significantly lower body weight, BMI and body length at 3 weeks as well as significantly lower body weight, BMI and waist circumference at 8 and 12 weeks were observed in newborn pups of IUGR rats compared with pups of control rats. Significant differences in ACho/H 2 O, ACr/H 2 O, AGlx/H 2 O and ALipid/H 2 O at 3 and 8 weeks as well as significant differences in ACr/H 2 O, ALipid/H 2 O and AGlx/H 2 O at 12 weeks were observed between pups of control rats and pups of IUGR rats. 1 H-MRS allows noninvasive assessment of liver metabolism in the rat and demonstrated the poor liver development of rats that experienced IUGR.

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes

PubMed Central

Kuga, Takahisa; Sasaki, Mitsuho; Mikami, Toshinari; Miake, Yasuo; Adachi, Jun; Shimizu, Maiko; Saito, Youhei; Koura, Minako; Takeda, Yasunori; Matsuda, Junichiro; Tomonaga, Takeshi; Nakayama, Yuji

2016-01-01

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts. PMID:27222304

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes.

PubMed

Kuga, Takahisa; Sasaki, Mitsuho; Mikami, Toshinari; Miake, Yasuo; Adachi, Jun; Shimizu, Maiko; Saito, Youhei; Koura, Minako; Takeda, Yasunori; Matsuda, Junichiro; Tomonaga, Takeshi; Nakayama, Yuji

2016-05-25

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.

Oxidative stress and cytotoxicity elicited lipid peroxidation in hemocytes of Bombyx mori larva infested with dipteran parasitoid, Exorista bombycis.

PubMed

Pooja, Makwana; Pradeep, Appukuttan Nair R; Hungund, Shambhavi P; Sagar, Chandrashekhar; Ponnuvel, Kangayam M; Awasthi, Arvind K; Trivedy, Kanika

2017-12-20

Parasitization of silkworm, Bombyx mori by invasive larva of dipteran parasitoid Exorista bombycis caused upto 20% revenue loss in sericulture. The parasitism was successful by suppressing host immune system however mechanism of immune suppression induced by E. bombycis is unknown which is unravelled here. The infestation induced cytotoxic symptoms in host hemocytes, such as vacuolated cytoplasm, porous plasma membrane, indented nuclei with condensed chromatin and dilated RER. One of the markers of necrosis is cell permeabilization, which can be measured as released lactate dehydrogenase (LDH). LDH level showed significantly (P<0.01) high release into extracellular medium in vitro after exposure of hemocytes to parasitoid larval tissue protein compared with control revealing membrane permeability and loss of cell integrity. At five minutes after exposure, cytotoxicity was 43% and was increased to 99% at 3h. The cytotoxicity is signalled by increased content of hydrogen peroxide (H2O2) causing lipid peroxidation followed by porosity in plasma membrane. A test for lipid peroxidation by measurement of lipid peroxidation breakdown product, malondialdehyde (MDA) revealed significant increase in peroxidation from one to 24 h post-invasion, with maximum at 12 h (P<0.008). Level of reactive oxygen species measured as H2O2 production increased from 6 to 12 h post-invasion and continued to increase significantly (P<0.03) reaching maximum at 48 h. These observations reveal that dipteran endoparasitoid invasion induced H2O2 production in the hemocytes causing cytotoxicity, lipid peroxidation and membrane porosity that suppressed both humoral- and cell-mediated immune responses of hemocytes in B. mori.

Pancreatic β-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide Species

NASA Astrophysics Data System (ADS)

Pilkington, Emily H.; Gurzov, Esteban N.; Kakinen, Aleksandr; Litwak, Sara A.; Stanley, William J.; Davis, Thomas P.; Ke, Pu Chun

2016-02-01

Aggregation of human islet amyloid polypeptide (hIAPP) into fibrils and plaques is associated with pancreatic β-cell loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hIAPP species is responsible for the observed toxicity and through what mechanisms remains ambiguous. In light of the importance of understanding hIAPP toxicity for T2D here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MIN6 cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. It has been found that all three hIAPP species, especially fresh hIAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers a new insight into β-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies of T2D as well as neurological disorders.

HSP90 is a therapeutic target in JAK2-dependent myeloproliferative neoplasms in mice and humans

PubMed Central

Marubayashi, Sachie; Koppikar, Priya; Taldone, Tony; Abdel-Wahab, Omar; West, Nathan; Bhagwat, Neha; Caldas-Lopes, Eloisi; Ross, Kenneth N.; Gönen, Mithat; Gozman, Alex; Ahn, James H.; Rodina, Anna; Ouerfelli, Ouathek; Yang, Guangbin; Hedvat, Cyrus; Bradner, James E.; Chiosis, Gabriela; Levine, Ross L.

2010-01-01

JAK2 kinase inhibitors were developed for the treatment of myeloproliferative neoplasms (MPNs), following the discovery of activating JAK2 mutations in the majority of patients with MPN. However, to date JAK2 inhibitor treatment has shown limited efficacy and apparent toxicities in clinical trials. We report here that an HSP90 inhibitor, PU-H71, demonstrated efficacy in cell line and mouse models of the MPN polycythemia vera (PV) and essential thrombocytosis (ET) by disrupting JAK2 protein stability. JAK2 physically associated with both HSP90 and PU-H71 and was degraded by PU-H71 treatment in vitro and in vivo, demonstrating that JAK2 is an HSP90 chaperone client. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth and signaling in JAK2 mutant cell lines and in primary MPN patient samples. PU-H71 treatment of mice resulted in JAK2 degradation, inhibition of JAK-STAT signaling, normalization of peripheral blood counts, and improved survival in MPN models at doses that did not degrade JAK2 in normal tissues or cause substantial toxicity. Importantly, PU-H71 treatment also reduced the mutant allele burden in mice. These data establish what we believe to be a novel therapeutic rationale for HSP90 inhibition in the treatment of JAK2-dependent MPN. PMID:20852385

Genomic analysis of lung cell lines exposures to space radiation and the effect of lunar dust on selected fibrosis gene using RT2 PCR Array

NASA Astrophysics Data System (ADS)

Yeshitla, Samrawit

In the United States (U.S.), lung cancer is the number one cause of cancer death among men and women. Previous studies on human and animal epithelial lung cells showed that ionizing radiation and certain environmental pollutants are carcinogens. The surface area of the lungs and the slow turnover rate of the epithelial cells are suggested to play a role in the vulnerability of the cells, which lead to increase in the progenitor cell of the lung. It has been proposed that these progenitor cells, when exposed to radiation undergo multiple alterations that cause the cells to become cancerous. The current thought is that the lungs contain several facultative progenitor cells that are situated throughout the lung epithelium and are regionally restricted in their regenerative capacity. In this study, normal Human Bronchial Epithelial Cells (HBECs) were immortalized through the expression of Cdk4 and hTERT and evaluated for the effects radiation using in vitro study. The HBECs retained its novel multipotent capacity in vitro and represented unrestricted progenitor cells of the adult lungs, which resemble an embryonic progenitor. Analysis of the transformed clones of human bronchial epithelial cell line, HEBC3KT exposed to Fe ions and gamma rays revealed chromosomal abnormality, which was detected with the Multi-color Fluorescent In Situ Hybridization (mFish). In Part two of this study the F344 rats exposed to lunar dust, for 4 weeks (6h/d; 5d/wk.) in nose-only inhalation chambers at concentrations of 0 (control air), 2.1, 6.8, 20.8, and 61 mg/m3 of lunar dust, were used to determine the lunar dust toxicity on the lung tissues and total RNA were prepared from the tissues and used for gene expression. Analysis of gene expression data using Ingenuity Pathway Analysis tool identified multiple pathways of which fibrosis was one of the pathways. The Rat Fibrosis RT 2 Profile PCR Array was used to profile the expression of 84 genes that are relevant to fibrosis in the lung tissue, after removing the infiltrated of the bronchoalveolar lavage (BAL) cells by lavaging. At a dose of 61mg/m 3, 29 genes showed changes and of 29 genes, nine genes, Bmp7, Cc112, Cc13, Itgb8, Serpinel, Smad6, Tgfbrl, Thbs2, and Tnf, showed significant fold change difference of up-regulation or down-regulation. The data in this study showed that iron ions and gamma rays promote chromosomal abnormality in HBEC3KT, and for the first time, the lunar dust altered gene expression of 29 genes that are relevant to fibrosis in the lung tissue.

EG-17SUV420-MEDIATED HETEROCHROMATIN CHANGES IN PEDIATRIC BRAIN CANCERS

PubMed Central

Van Meter, Timothy E.; Terry, Jocelyn; Rockwell, Nathan; Goggin, Sarah; Nethala, Priya; Khan, Asadullah

2014-01-01

Silencing mechanisms play a role in genomic stability by maintaining condensed, non-active regions of the genome. SUV420 enzymes contain a SET domain conferring methyltransferase activity toward histones. The Histone H4 lysine 20 trimethylation (H4K20me3) mark maintained by SUV420H2 is associated with heterochromatin formation and gene silencing, whereas the dimethylated mark (H4K20me2) is associated with DNA repair. In studies of epigenetic factors in large patient cohorts with ependymoma, it was found that SUV420H2 expression was lost or diminished in patients with reciprocal increases in prognostic markers such as hTERT. To better understand the normal function of Suv4-20H1/H2 enzyme in neural progenitors, and pathological changes in cancers, a variety of differentiation paradigms were used. The NT2D1 neurally restricted cell line, and BGO1V and H9 human embryonic stem cells (ESCs), and differentiated progeny, were used alongside tumors to better understand enzyme targets and functional outcomes (e.g.,lineage, differentiation, regional chromatin modifications). Lineage stages were verified with stage-specific markers by immunofluorescence and qPCR. Suv4-20 H1 and H2 were present in ESCs and neural progenitors and decreased thereafter. RNAi knockdown of SUV420 enzymes led to decreased H4K20 methylation in cancer cells. DNA methylation microarrays and ChIP-PCR suggest 1) that SUV420 is not regulated by DNA methylation in ependymomas; 2) that active chromatin marks such as H3K4 dimethylation are enriched near the transcriptional start site in the SUV420H2 gene, and 3) that hTERT is hyper-methylated at specific CpG islands and histones in a tumor sub-group-specific manner. This data supports the hypothesis that Suv4-20H2 is highly active in progenitor cells and functionally lost in some brain cancers. These studies begin to elucidate coincident mechanisms of gene silencing active in neural progenitors that may be altered in a subset of pediatric brain cancers.

O2⋅- and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate.

PubMed

Schoenfeld, Joshua D; Sibenaller, Zita A; Mapuskar, Kranti A; Wagner, Brett A; Cramer-Morales, Kimberly L; Furqan, Muhammad; Sandhu, Sonia; Carlisle, Thomas L; Smith, Mark C; Abu Hejleh, Taher; Berg, Daniel J; Zhang, Jun; Keech, John; Parekh, Kalpaj R; Bhatia, Sudershan; Monga, Varun; Bodeker, Kellie L; Ahmann, Logan; Vollstedt, Sandy; Brown, Heather; Shanahan Kauffman, Erin P; Schall, Mary E; Hohl, Ray J; Clamon, Gerald H; Greenlee, Jeremy D; Howard, Matthew A; Schultz, Michael K; Smith, Brian J; Riley, Dennis P; Domann, Frederick E; Cullen, Joseph J; Buettner, Garry R; Buatti, John M; Spitz, Douglas R; Allen, Bryan G

2017-04-10

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H 2 O 2 ; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O 2 ⋅- and H 2 O 2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H 2 O 2 . In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of H2 photoproduction by marine green alga Tetraselmis subcordiformis integrated with an alkaline fuel cell.

PubMed

Guo, Zhen; Li, Ying; Guo, Haiyan

2016-03-01

To investigate the feasibility of coupling carbonyl cyanide m-chlorophenylhydrazone-regulated photohydrogen production by Tetraselmis subcordiformis in a photobioreactor to an alkaline fuel cell (AFC). H2 evolution kinetics in the AFC integrated process was characterized. The duration of H2 evolution was prolonged and its yield was improved about 1.5-fold (to 78 ± 5 ml l(-1)) compared with that of the process without AFC. Improved H2 yield was possibly caused by removal of H2 feedback inhibition by H2 consumption in situ. Decreases in the H2 production rate correlated with the gradual deactivation of PSII and hydrogenase activities. The H2 yield was closely associated with catabolism of starch and protein. A marine green algal CO2-supplemented culture integrated with in situ H2-consumption by an AFC system was developed as a viable protocol for the H2 production.

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells.

PubMed

Matsunaga, Yusuke; Fukuma, Daiki; Hirata, Shinya; Fukushima, Satoshi; Haruta, Miwa; Ikeda, Tokunori; Negishi, Izumi; Nishimura, Yasuharu; Senju, Satoru

2008-11-01

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

6-Mercaptopurine (6-MP) induces cell cycle arrest and apoptosis of neural progenitor cells in the developing fetal rat brain.

PubMed

Kanemitsu, H; Yamauchi, H; Komatsu, M; Yamamoto, S; Okazaki, S; Uchida, K; Nakayama, H

2009-01-01

6-Mercaptopurine (6-MP), an analogue of hypoxanthine, is used in the therapy of acute lymphoblastic leukemia and causes fetal neurotoxicity. To clarify the mechanisms of 6-MP-induced fetal neurotoxicity leading to the cell cycle arrest and apoptosis of neural progenitor cells, pregnant rats were treated with 50 mg/kg 6-MP on embryonic day (E) 13, and the fetal telencephalons were examined at 12 to 72 h (h) after treatment. Flow-cytometric analysis confirmed an accumulation of cells at G2/M, S, and sub-G1 (apoptotic cells) phases from 24 to 72 h. The number of phosphorylated histone H3-positive cells (mitotic cells) decreased from 36 to 72 h, and the phosphorylated (active) form of p53 protein, which is a mediator of apoptosis and cell cycle arrest, increased from 24 to 48 h. An executor of p53-mediated cell cycle arrest, p21, showed intense overexpression at both the mRNA and protein levels from 24 to 72 h. Cdc25A protein, which is needed for the progression of S phase, decreased at 36 and 48 h. In addition, phosphorylated cdc2 protein, which is an inactive form of cdc2 necessary for G2/M progression, increased from 24 to 48 h. These results suggest that 6-MP induced G2/M arrest, delayed S-phase progression, and finally induced apoptosis of neural progenitor cells mediated by p53 in the fetal rat telencephalon.

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

2011-01-21

Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

RGS2 is regulated by angiotensin II and functions as a negative feedback of aldosterone production in H295R human adrenocortical cells.

PubMed

Romero, Damian G; Plonczynski, Maria W; Gomez-Sanchez, Elise P; Yanes, Licy L; Gomez-Sanchez, Celso E

2006-08-01

Regulator of G protein signaling (RGS) proteins interact with Galpha-subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. Angiotensin (Ang) II interacts with its G protein-coupled receptor in zona glomerulosa adrenal cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. We studied Ang II-mediated regulation of RGS2, the role of RGS2 in steroidogenesis, and the intracellular signal events involved in H295R human adrenal cells. We report that both H295R cells and human adrenal gland express RGS2 mRNA. In H295R cells, Ang II caused a rapid and transient increase in RGS2 mRNA levels quantified by real-time RT-PCR. Ang II effects were mimicked by calcium ionophore A23187 and blocked by calcium channel blocker nifedipine. Ang II effects also were blocked by calmodulin antagonists (W-7 and calmidazolium) and calcium/calmodulin-dependent kinase antagonist KN-93. RGS2 overexpression by retroviral infection in H295R cells caused a decrease in Ang II-stimulated aldosterone secretion but did not modify cortisol secretion. In reporter assays, RGS2 decreased Ang II-mediated aldosterone synthase up-regulation. These results suggest that Ang II up-regulates RGS2 mRNA by the calcium/calmodulin-dependent kinase pathway in H295R cells. RGS2 overexpression specifically decreases aldosterone secretion through a decrease in Ang II-mediated aldosterone synthase-induced expression. In conclusion, RGS2 expression is induced by Ang II to terminate the intracellular signaling cascade generated by Ang II. RGS2 alterations in expression levels or functionality could be implicated in deregulations of Ang II signaling and abnormal aldosterone secretion by the adrenal gland.

Revelation of the dynamic progression of hypoxia-reoxygenation injury by visualization of the lysosomal hydrogen peroxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Yuanjun; Zhou, Tongliang; Yang, Lingfei

Hydrogen peroxide (H{sub 2}O{sub 2}) plays an important role in pathological conditions, such as cerebral ischemia-reperfusion (I-R) injury. Fluorescent probes may serve as valuable tools to detect the amount, temporal and spatial distribution of H{sub 2}O{sub 2} in living cells. To investigate the role of lysosomal H{sub 2}O{sub 2} involved in cerebral I-R injury, we designed and synthesized a lysosome-targetable two-photon fluorescent probe ztl-4, through expansion and substitution of the original pyridazinone scaffold, conjugation of electronic-donating aromatic ring and precise terminal modification of the alkyl linker. The probe ztl-4 exhibited fast, sensitive and highly selective response toward H{sub 2}O{sub 2}.more » ztl-4 could image exogenous H{sub 2}O{sub 2} in SH-SY5Y cells and brain slices. In addition, ztl-4 was located in lysosomes with high colocalization coefficient compared with LysoTracker. ztl-4 was further applied for detecting the endogenous generation of H{sub 2}O{sub 2} in SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) or OGD/reoxygenation (OGD/R) injury. Both OGD- and OGD/R-induced cell injury caused a time-dependent increase of H{sub 2}O{sub 2} production within lysosomes. Moreover, OGD/R-treated cells showed much more amount of H{sub 2}O{sub 2} than OGD-treated cells, indicating that reoxygenation will promote H{sub 2}O{sub 2} accumulation in lysosomes of post-hypoxia cells. Therefore, the probe is suitable for monitoring the dynamic changes of lysosomal H{sub 2}O{sub 2} in cells. - Highlights: • New fluorescent probe displays high selectivity for H{sub 2}O{sub 2}. • The probe is lysosome-targetable. • The probe can real-time monitor hypoxia/reoxygenation injury-induced generation of H{sub 2}O{sub 2} in lysosomes of cells.« less

Atrazine activates multiple signaling pathways enhancing the rapid hCG-induced androgenesis in rat Leydig cells.

PubMed

Pogrmic-Majkic, Kristina; Fa, Svetlana; Samardzija, Dragana; Hrubik, Jelena; Kaisarevic, Sonja; Andric, Nebojsa

2016-08-10

Atrazine (ATR) is an endocrine disruptor that affects steroidogenic process, resulting in disruption of reproductive function of the male and female gonads. In this study, we used the primary culture of peripubertal Leydig cells to investigate the effect of ATR on the rapid androgen production stimulated by human chorionic gonadotropin (hCG). We demonstrated that ATR activated multiple signaling pathways enhancing the rapid hCG-stimulated androgen biosynthesis in Leydig cells. Low hCG concentration (0.25ng/mL) caused cAMP-independent, but ERK1/2-dependent increase in androgen production after 60min of incubation. Co-treatment with ATR for 60min enhanced the cAMP production in hCG-stimulated cells. Accumulation of androgens was prevented by addition of U0126, N-acetyl-l-cysteine and AG1478. Co-treatment with hCG and ATR for 60min did not alter steroidogenic acute regulatory protein (Star) mRNA level in Leydig cells. After 120min, hCG further increased androgenesis in Leydig cells that was sensitive to inhibition of the cAMP/PKA, ERK1/2 and ROS signaling pathways. Co-treatment with ATR for 120min further enhanced the hCG-induced androgen production, which was prevented by inhibition of the calcium, PKC and EGFR signaling cascades. After 120min, ATR enhanced the expression of Star mRNA in hCG-stimulated Leydig cells through activation of the PKA and PKC pathway. Collectively, these data suggest that exposure to ATR caused perturbations in multiple signaling pathways, thus enhancing the rapid hCG-dependent androgen biosynthesis in peripubertal Leydig cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Human T-cells directed to seasonal influenza A virus cross-react with 2009 pandemic influenza A (H1N1) and swine-origin triple-reassortant H3N2 influenza viruses.

PubMed

Hillaire, Marine L B; Vogelzang-van Trierum, Stella E; Kreijtz, Joost H C M; de Mutsert, Gerrie; Fouchier, Ron A M; Osterhaus, Albert D M E; Rimmelzwaan, Guus F

2013-03-01

Virus-specific CD8(+) T-cells contribute to protective immunity against influenza A virus (IAV) infections. As the majority of these cells are directed to conserved viral proteins, they may afford protection against IAVs of various subtypes. The present study assessed the cross-reactivity of human CD8(+) T-lymphocytes, induced by infection with seasonal A (H1N1) or A (H3N2) influenza virus, with 2009 pandemic influenza A (H1N1) virus [A(H1N1)pdm09] and swine-origin triple-reassortant A (H3N2) [A(H3N2)v] viruses that are currently causing an increasing number of human cases in the USA. It was demonstrated that CD8(+) T-cells induced after seasonal IAV infections exerted lytic activity and produced gamma interferon upon in vitro restimulation with A(H1N1)pdm09 and A(H3N2)v influenza A viruses. Furthermore, CD8(+) T-cells directed to A(H1N1)pdm09 virus displayed a high degree of cross-reactivity with A(H3N2)v viruses. It was concluded that cross-reacting T-cells had the potential to afford protective immunity against A(H1N1)pdm09 viruses during the pandemic and offer some degree of protection against infection with A(H3N2)v viruses.

Hydrogen gas alleviates oxygen toxicity by reducing hydroxyl radical levels in PC12 cells.

PubMed

Yu, Junchao; Yu, Qiuhong; Liu, Yaling; Zhang, Ruiyun; Xue, Lianbi

2017-01-01

Hyperbaric oxygen (HBO) therapy through breathing oxygen at the pressure of above 1 atmosphere absolute (ATA) is useful for varieties of clinical conditions, especially hypoxic-ischemic diseases. Because of generation of reactive oxygen species (ROS), breathing oxygen gas at high pressures can cause oxygen toxicity in the central nervous system, leading to multiple neurological dysfunction, which limits the use of HBO therapy. Studies have shown that Hydrogen gas (H2) can diminish oxidative stress and effectively reduce active ROS associated with diseases. However, the effect of H2 on ROS generated from HBO therapy remains unclear. In this study, we investigated the effect of H2 on ROS during HBO therapy using PC12 cells. PC12 cells cultured in medium were exposed to oxygen gas or mixed oxygen gas and H2 at 1 ATA or 5 ATA. Cells viability and oxidation products and ROS were determined. The data showed that H2 promoted the cell viability and inhibited the damage in the cell and mitochondria membrane, reduced the levels of lipid peroxidation and DNA oxidation, and selectively decreased the levels of •OH but not disturbing the levels of O2•-, H2O2, or NO• in PC12 cells during HBO therapy. These results indicated that H2 effectively reduced •OH, protected cells against oxygen toxicity resulting from HBO therapy, and had no effect on other ROS. Our data supported that H2 could be potentially used as an antioxidant during HBO therapy.

MPK6 controls H2 O2-induced root elongation by mediating Ca2+ influx across the plasma membrane of root cells in Arabidopsis seedlings.

PubMed

Han, Shuan; Fang, Lin; Ren, Xuejian; Wang, Wenle; Jiang, Jing

2015-01-01

Mitogen-activated protein kinases (MPKs) play critical roles in signalling and growth, and Ca(2+) and H2 O2 control plant growth processes associated with abscisic acid (ABA). However, it remains unclear how MPKs are involved in H2 O2 - and Ca(2+) -mediated root elongation. Root elongation in seedlings of the loss-of-function mutant Atmpk6 (Arabidopsis thaliana MPK6) was less sensitive to moderate H2 O2 or ABA than that in wild-type (WT) plants. The enhanced elongation was a result of root cell expansion. This effect disappeared when ABA-induced H2 O2 accumulation or the cytosolic Ca(2+) increase were defective. Molecular and biochemical evidence showed that increased expression of the cell wall peroxidase PRX34 in Atmpk6 root cells enhanced apoplastic H2 O2 generation; this promoted a cytosolic Ca(2+) increase and Ca(2+) influx across the plasma membrane. The plasma membrane damage caused by high levels of H2 O2 was ameliorated in a Ca(2+) -dependent manner. These results suggested that there was intensified PRX34-mediated H2 O2 generation in the apoplast and increased Ca(2+) flux into the cytosol of Atmpk6 root cells; that is, the spatial separation of apoplastic H2 O2 from cytosolic Ca(2+) in root cells prevented H2 O2 -induced inhibition of root elongation in Atmpk6 seedlings. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

T Cells that Recognize HPV Protein Can Target Virus-Infected Cells | Center for Cancer Research

Cancer.gov

Treating patients with T cells isolated from a tumor and subsequently expanded in the lab can cause the complete regression of some melanomas and cervical cancers, but the treatment is currently restricted to a few cancer types.

Effect of pH and glucose on cultured human peritoneal mesothelial cells.

PubMed

Shao, J C; Yorioka, N; Nishida, Y; Yamakido, M

1999-08-01

We investigated the effects of various pH and glucose concentrations on the growth of human peritoneal mesothelial cells and on coagulation and fibrinolytic factors. Cells were cultured at various pH values in Ham's F-12 medium containing 1.0% foetal calf serum and supplemented with D-glucose or D-mannitol at various concentrations. After 4-48 h, cell proliferation and 3H-thymidine incorporation were determined. Coagulation and fibrinolytic factors were measured after 48 h. Glucose caused concentration-dependent inhibition of cell growth at all pH values, but the deleterious effect of low pH on cell proliferation was faster and stronger than that of high glucose. At a similar osmolality, mannitol caused less inhibition of cell proliferation than glucose. There was a glucose concentration-dependent increase of thrombin-antithrombin III complex production at all pH values. At pH 5.2, tissue-type plasminogen activator production was far lower than at higher pH values, and production of the plasminogen activator inhibitor showed a glucose concentration-dependent increase. At pH 6.5 or 7.3, however, the plasminogen activator inhibitor production decreased and tissue-type plasminogen activator production increased in a glucose concentration-dependent manner. Low pH and/or high glucose culture medium had an inhibitory effect on peritoneal mesothelial cells, with the effect of high glucose being partially related to hyperosmolality. These cells may modulate peritoneal coagulant and fibrinolytic activity, with the balance between coagulation and fibrinolysis being disturbed by low pH and/or high glucose.

Cell biology of sarcomeric protein engineering: disease modeling and therapeutic potential.

PubMed

Thompson, Brian R; Metzger, Joseph M

2014-09-01

The cardiac sarcomere is the functional unit for myocyte contraction. Ordered arrays of sarcomeric proteins, held in stoichiometric balance with each other, respond to calcium to coordinate contraction and relaxation of the heart. Altered sarcomeric structure-function underlies the primary basis of disease in multiple acquired and inherited heart disease states. Hypertrophic and restrictive cardiomyopathies are caused by inherited mutations in sarcomeric genes and result in altered contractility. Ischemia-mediated acidosis directly alters sarcomere function resulting in decreased contractility. In this review, we highlight the use of acute genetic engineering of adult cardiac myocytes through stoichiometric replacement of sarcomeric proteins in these disease states with particular focus on cardiac troponin I. Stoichiometric replacement of disease causing mutations has been instrumental in defining the molecular mechanisms of hypertrophic and restrictive cardiomyopathy in a cellular context. In addition, taking advantage of stoichiometric replacement through gene therapy is discussed, highlighting the ischemia-resistant histidine-button, A164H cTnI. Stoichiometric replacement of sarcomeric proteins offers a potential gene therapy avenue to replace mutant proteins, alter sarcomeric responses to pathophysiologic insults, or neutralize altered sarcomeric function in disease. © 2014 Wiley Periodicals, Inc.

Investigation into the effects of sulfur on syngas reforming inside a solid oxide fuel cell

NASA Astrophysics Data System (ADS)

Li, Ting Shuai; Xu, Min; Gao, Chongxin; Wang, Baoqing; Liu, Xiyun; Li, Baihai; Wang, Wei Guo

2014-07-01

The electrochemical performance and long-term durability of a solid oxide fuel cell have been evaluated with a simulated coal syngas containing 2 ppm H2S as fuel. The resulting impedance spectra indicate that no observable power loss is caused by the addition of 2 ppm H2S, and the cell shows stability of nearly 500 h at 0.625 A cm-2. The composition of mixed gas is analyzed both at a current load of 0.625 A cm-2 and open circuit state. Hydrogen and carbon monoxide are directly consumed as fuels at the anode side, whereas methane stays unchanged during the operation. It seems the internal carbohydrate reforming and impurity poisoning interacts and weakens the poisoning effects. The oxidation of H2 and the water gas shift reaction take advantages over methane reforming at the cell operational conditions.

A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: implications for tumorigenesis

PubMed Central

Craig, Errol; Zhang, Zhi-Kai; Davies, Kelvin P.; Kalpana, Ganjam V.

2002-01-01

INI1 (integrase interactor 1)/hSNF5 is a component of the mammalian SWI/SNF complex and a tumor suppressor mutated in malignant rhabdoid tumors (MRT). We have identified a nuclear export signal (NES) in the highly conserved repeat 2 domain of INI1 that is unmasked upon deletion of a downstream sequence. Mutation of conserved hydrophobic residues within the NES, as well as leptomycin B treatment abrogated the nuclear export. Full-length INI1 specifically associated with hCRM1/exportin1 in vivo and in vitro. A mutant INI1 [INI1(1–319) delG950] found in MRT lacking the 66 C-terminal amino acids mislocalized to the cytoplasm. Full-length INI1 but not the INI1(1–319 delG950) mutant caused flat cell formation and cell cycle arrest in cell lines derived from MRT. Disruption of the NES in the delG950 mutant caused nuclear localization of the protein and restored its ability to cause cell cycle arrest. These observations demonstrate that INI1 has a masked NES that mediates regulated hCRM1/exportin1-dependent nuclear export and we propose that mutations that cause deregulated nuclear export of the protein could lead to tumorigenesis. PMID:11782423

Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells

PubMed Central

Simonis, Alexander; Hebling, Sabrina; Gulbins, Erich; Schneider-Schaulies, Sibylle; Schubert-Unkmeir, Alexandra

2014-01-01

The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. PMID:24945304

Modeling the chemical kinetics of atmospheric plasma for cell treatment in a liquid solution

NASA Astrophysics Data System (ADS)

Kim, H. Y.; Lee, H. W.; Kang, S. K.; Wk. Lee, H.; Kim, G. C.; Lee, J. K.

2012-07-01

Low temperature atmospheric pressure plasmas have been known to be effective for living cell inactivation in a liquid solution but it is not clear yet which species are key factors for the cell treatment. Using a global model, we elucidate the processes through which pH level in the solution is changed from neutral to acidic after plasma exposure and key components with pH and air variation. First, pH level in a liquid solution is changed by He+ and He(21S) radicals. Second, O3 density decreases as pH level in the solution decreases and air concentration decreases. It can be a method of removing O3 that causes chest pain and damages lung tissue when the density is very high. H2O2, HO2, and NO radicals are found to be key factors for cell inactivation in the solution with pH and air variation.

High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells

EPA Science Inventory

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2,060 chemical samples...

H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response.

PubMed

Levine, A; Tenhaken, R; Dixon, R; Lamb, C

1994-11-18

Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. We report here that H2O2 from this oxidative burst not only drives the cross-linking of cell wall structural proteins, but also functions as a local trigger of programmed death in challenged cells and as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. Thus, H2O2 from the oxidative burst plays a key role in the orchestration of a localized hypersensitive response during the expression of plant disease resistance.

Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

PubMed Central

Kwun, Hyun Jin; Shuda, Masahiro; Camacho, Carlos J.; Gamper, Armin M.; Thant, Mamie; Chang, Yuan

2015-01-01

ABSTRACT Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP), including PP2A A and C subunits and PP4C, as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of nonhuman polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT coimmunopurified with subunits PP2A Aα and PP2A C, MCV sT coimmunopurified with PP2A Aα, PP2A Aβ, and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV sT protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56α into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55α, B56α and B56ε into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth, but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting. IMPORTANCE Merkel cell polyomavirus is a newly discovered human cancer virus that promotes cancer, in part, through expression of its small T (sT) oncoprotein. Animal polyomavirus sT oncoproteins have been found to cause experimental tumors by blocking the activities of a group of phosphatases called protein phosphatase 2A (PP2A). Our structural analysis reveals that MCV sT also displaces the B subunit of PP2A to inhibit PP2A activity. MCV sT, however, only displaces a restricted subset of PP2A B subunits, which is insufficient to cause tumor cell formation in vitro. MCV sT instead transforms tumor cells through another region called the large T stabilization domain. The PP2A targeting and transforming activities lie on opposite faces of the MCV sT molecule and can be genetically separated from each other. PMID:25631078

Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system.

PubMed

Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Dohda, Takeaki; Kino-Oka, Masahiro

2018-03-01

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k L a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h -1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 10 9 cells L -1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018. © 2017 American Institute of Chemical Engineers.

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

PubMed Central

Wynford-Thomas, D; Bond, J A; Wyllie, F S; Burns, J S; Williams, E D; Jones, T; Sheer, D; Lemoine, N R

1990-01-01

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types. Images PMID:1697930

Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

PubMed

Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

2016-05-01

Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

PubMed Central

van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

2012-01-01

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699

EphA2 modulates radiosensitive of hepatocellular carcinoma cells via p38/mitogen-activated protein kinase-mediated signal pathways.

PubMed

Jin, Qiao; Li, Xiangjun; Cao, Peiguo

2015-10-01

This experiment was conducted to investigate the role of EPH receptor A2 (EphA2) in the modulation of radiosensitivity of hepatic cellular cancer (HCC) cells and to determine whether p38/mitogen-activated protein kinase (p38MAPK) signaling mediated EphA2 function in this respect. The protein expressions of EphA2 and phosphorylated p38MAPK were tested in HCC and normal hepatic tissues. In HCC 97H cells, EphA2 was overexpressed and knocked out by transfection with EphA2 expression vector and EphA2-ShRNA, respectively, prior to cell exposure to low-dose irradiation. Significantly upregulated EphA2 and phosphorylated p38MAPK were observed in HCC tissues, compared with those in normal hepatic tissues. Low-dose irradiation (1 Gy) only caused minor damage to HCC 97H cells, as assessed by alterations in cell viability, apoptosis rate, and cell healing capacity (p = 0.072, p = 0.078, and p = 0.069 respectively). However, EphA2 knock-out in HCC 97H cells induced significant reduction in cell viability and cell healing capacity after these cells were subjected to low-dose irradiation. Apoptosis rate underwent dramatic increase (p < 0.01). By contrast, EphA2 overexpression in HCC 97H cells reversed these effects and enhanced cell colony formation rate, thus displaying remarkable attenuation of radiosensitivity of HCC 97H cells. Further, SB203580, a specific inhibitor of p38MAPK, was added to HCC 97H cells over-expressing EphA2. The effect of EphA2 overexpression on the radiosensitivity of HCC 97H cells was abrogated. Thus, the present study indicates that EphA2 have the ability to negatively regulate the radiosensitivity of HCC 97H cells, which mainly depends on 38MAPK-mediated signal pathways. Copyright © 2015. Published by Elsevier Taiwan.

Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation.

PubMed

Gröschl, Benedikt; Bettstetter, Marcus; Giedl, Christian; Woenckhaus, Matthias; Edmonston, Tina; Hofstädter, Ferdinand; Dietmaier, Wolfgang

2013-04-01

DUSP4 (MKP-2), a member of the mitogen-activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen-activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI-H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI-H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT-qPCR. DUSP4 was overexpressed in all 19 MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p < 0.001). Consistently, MSI-H CRC cell lines showed 6.8-fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC. Copyright © 2012 UICC.

Altered Viral Replication and Cell Responses by Inserting MicroRNA Recognition Element into PB1 in Pandemic Influenza A Virus (H1N1) 2009

PubMed Central

Shen, Xiaoyue; Sun, Wenkui; Shi, Yi; Xing, Zheng; Su, Xin

2015-01-01

Objective. MicroRNAs (miRNAs) are endogenous noncoding RNAs that spatiotemporally modulate mRNAs in a posttranscriptional manner. Engineering mutant viruses by inserting cell-specific miRNA recognition element (MRE) into viral genome may alter viral infectivity and host responses in vital tissues and organs infected with pandemic influenza A virus (H1N1) 2009 (H1N1pdm). Methods. In this study, we employed reverse genetics approach to generate a recombinant H1N1pdm with a cell-specific miRNA target sequence inserted into its PB1 genomic segment to investigate whether miRNAs are able to suppress H1N1pdm replication. We inserted an MRE of microRNA-let-7b (miR-let-7b) into the open reading frame of PB1 to test the feasibility of creating a cell-restricted H1N1pdm virus since let-7b is abundant in human bronchial epithelial cells. Results. miR-let-7b is rich in human bronchial epithelial cells (HBE). Incorporation of the miR-let-7b-MRE confers upon the recombinant H1N1pdm virus susceptibility to miR-let-7b targeting, suggesting that the H1N1pdm and influenza A viruses can be engineered to exert the desired replication restrictive effect and decrease infectivity in vital tissues and organs. Conclusions. This approach provides an additional layer of biosafety and thus has great potential for the application in the rational development of safer and more effective influenza viral vaccines. PMID:25788763

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

PubMed Central

Kelton, William; Waindok, Ann Cathrin; Pesch, Theresa; Pogson, Mark; Ford, Kyle; Parola, Cristina; Reddy, Sai T.

2017-01-01

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation. PMID:28374766

Neuroligin-3 protects retinal cells from H2O2-induced cell death via activation of Nrf2 signaling.

PubMed

Li, Xiu-Miao; Huang, Dan; Yu, Qing; Yang, Jian; Yao, Jin

2018-05-25

Intensified oxidative stress can cause severe damage to human retinal pigment epithelium (RPE) cells and retinal ganglion cells (RGCs). The potential effect of neuroligin-3 (NLGN3) against the process is studied here. Our results show that NLGN3 efficiently inhibited hydrogen peroxide (H 2 O 2 )-induced death and apoptosis in human RPE cells and RGCs. H 2 O 2 -induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage in retinal cells were alleviated by NLGN3. NLGN3 activated nuclear-factor-E2-related factor 2 (Nrf2) signaling, enabling Nrf2 protein stabilization, nuclear translocation and expression of key anti-oxidant enzymes (HO1, NOQ1 and GCLC) in RPE cells and RGCs. Further results demonstrate that NLGN3 activated Akt-mTORC1 signaling in retinal cells. Conversely, Akt-mTORC1 inhibitors (RAD001 and LY294002) reduced NLGN3-induced HO1, NOQ1 and GCLC mRNA expression. Significantly, Nrf2 silencing by targeted shRNAs reversed NLGN3-induced retinal cytoprotection against H 2 O 2 . We conclude that NLGN3 activates Nrf2 signaling to protect human retinal cells from H 2 O 2 . NLGN3 could be further tested as a valuable retinal protection agent. Copyright © 2018 Elsevier Inc. All rights reserved.

Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in Pisum sativum.

PubMed

Puli, Mallikarjuna Rao; Rajsheel, Pidakala; Aswani, Vetcha; Agurla, Srinivas; Kuchitsu, Kazuyuki; Raghavendra, Agepati S

2016-10-01

Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N',N'-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

Restriction point control of cell growth by a labile protein: evidence for increased stability in transformed cells.

PubMed Central

Campisi, J; Medrano, E E; Morreo, G; Pardee, A B

1982-01-01

It has been proposed that animal cells must accumulate a labile protein(s) before they can pass the restriction (R) point in the G1 phase of the cell cycle [Rossow, P. W., Riddle, V. G. H. & Pardee, A. B. (1979) Proc. Natl. Acad. Sci. USA 76, 4446--4450]. Here, we present evidence that this R protein acquires increased stability in transformed 3T3 cells, thereby allowing these cells to continue growth under conditions that arrest untransformed cells. Low doses of cycloheximide or histidinol drastically reduced the rate at which normal 3T3 (A31) fibroblasts in early G1 could enter DNA synthesis. These drugs had less effect on entry of two tumorigenic A31 derivatives, BPA31 and SVA31, in S, although measurement of [3H]leucine incorporation showed that the inhibitors were equally effective in the three cell lines. The hypothesis is that the transformed lines are less sensitive because moderate inhibition of their R protein synthesis is compensated by lower rates of protein degradation. To test this idea, we completely inhibited cytoplasmic protein synthesis for several hours shortly before A31 and BPA31 cells had reached the R point. After removal of inhibitor, A31 cells showed delays in the onset of S that were in excess of the inhibitor pulse, consistent with decay of labile protein during the pulse. BPA31 cells showed no excess delays, suggesting a much more stable R protein. The half-life of the R protein was estimated as 2.5 hr in A31 cells, indicating that, in these cells, R protein synthesis starts at the beginning of G1. In the BPA31 cells the R protein showed no signs of decay for at least 8 hr. PMID:6952194

Telomere Restriction Fragment (TRF) Analysis.

PubMed

Mender, Ilgen; Shay, Jerry W

2015-11-20

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al. , 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells. Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al. , 1990; Ouellette et al. , 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Effects of immobilisation and caloric restriction on antioxidant parameters and T-cell apoptosis in healthy young men

NASA Astrophysics Data System (ADS)

Ellinger, S.; Arendt, B. M.; Boese, A.; Juschus, M.; Schaefer, S.; Stoffel-Wagner, B.; Goerlich, R.

Background: Astronauts are exposed to oxidative stress due to radiation and microgravity, which might impair immune functions. Effects of hypocaloric nutrition as often observed in astronauts on oxidative stress and immune functions are not clear. We investigated, if microgravity, simulated by 6 Head-down tilt (HDT) and caloric restriction (-25%, fat reduced) with adequate supply of micronutrients affect DNA-damage in peripheral leukocytes, antioxidant parameters in plasma, and T-cell apoptosis. Material & Methods: 10 healthy male non-smokers were subjected to 4 different interventions (normocaloric diet or caloric restriction (CR) in upright position (UP) or HDT) for 14 days each (cross-over). DNA-damage in peripheral leukocytes (Comet Assay), trolox equivalent antioxidant capacity (TEAC) and uric acid in plasma were measured before, after 5, 10, and 13 days of intervention, and after 2 days recovery. T-cell apoptosis (Annexin V binding test) was assessed before and after intervention. Results: Preliminary results show that only endogenous, but not ex vivo H2O2-induced DNA strand breaks were reduced by CR compared to normocaloric diet. In upright position, endogenous DNA strand breaks decreased continuously during CR, reaching significance after recovery. During HDT, caloric restriction seems to counteract a temporary increase in DNA strand breaks observed in subjects receiving normocaloric diet. TEAC was reduced during HDT compared to UP in subjects under caloric restriction. An increase in plasma uric acid related to intervention occurred only after 5 days HDT in CR vs. normocaloric diet. T-cell apoptosis was not affected by any kind of intervention. Conclusion: Neither HDT nor CR with sufficient supply of micronutrients seem to induce oxidative stress or T-cell apoptosis in healthy young men. In contrast, CR might prevent endogenous DNA-damage in peripheral leukocytes. As DNA-damage is a risk factor for carcinogenesis, protective effects of energy reduction are worth being investigated in future studies. Acknowledgement: This work was supported by the European Space Agency (AO-LS-2000-BR-SHORT-007) and the German Federal Ministry of Education and Research (BMBF) (55 WB 0155).

Attenuation of Oxidative Stress-Induced Cell Apoptosis in Schwann RSC96 Cells by Ocimum Gratissimum Aqueous Extract

PubMed Central

Chao, Pei-Yu; Lin, James A.; Ye, Je-Chiuan; Hwang, Jin-Ming; Ting, Wei-Jen; Huang, Chih-Yang; Liu, Jer-Yuh

2017-01-01

Objectives:Cell transplantation therapy of Schwann cells (SCs) is a promising therapeutic strategy after spinal cord injury. However, challenges such as oxidative stress hinder satisfactory cell viability and intervention for enhancing SCs survival is critical throughout the transplantation procedures. Ocimum gratissimum, widely used as a folk medicine in many countries, has therapeutic and anti-oxidative properties and may protect SCs survival. Methods:We examined the protective effects of aqueous O. gratissimum extract (OGE) against cell damage caused by H2O2-induced oxidative stress in RSC96 Schwann cells. Results:Our results showed that the RSC96 cells, damaged by H2O2 oxidative stress, decreased their viability up to 32% after treatment with different concentrations of up to 300 μM H2O2, but OGE pretreatment (150 or 200 μg/mL) increased cell viability by approximately 62% or 66%, respectively. Cell cycle analysis indicated a high (43%) sub-G1 cell population in the H2O2-treated RSC96 cells compared with untreated cells (1%); whereas OGE pretreatment (150 and 200 μg/mL) of RSC96 cells significantly reduced the sub-G1 cells (7% and 8%, respectively). Furthermore, Western blot analysis revealed that OGE pretreatment inhibited H2O2-induced apoptotic protein caspase-3 activation and PARP cleavage, as well as it reversed Bax up-regulation and Bcl-2 down-regulation. The amelioration of OGE of cell stress and stress-induced apoptosis was proved by the HSP70 and HSP72 decrease. Conclusion: Our data suggest that OGE may minimize the cytotoxic effects of H2O2-induced SCs apoptosis by modulating the apoptotic pathway and could potentially supplement cell transplantation therapy. PMID:28824312

Calorie Restriction Attenuates Terminal Differentiation of Immune Cells.

PubMed

White, Matthew J; Beaver, Charlotte M; Goodier, Martin R; Bottomley, Christian; Nielsen, Carolyn M; Wolf, Asia-Sophia F M; Boldrin, Luisa; Whitmore, Charlotte; Morgan, Jennifer; Pearce, Daniel J; Riley, Eleanor M

2016-01-01

Immune senescence is a natural consequence of aging and may contribute to frailty and loss of homeostasis in later life. Calorie restriction increases healthy life-span in C57BL/6J (but not DBA/2J) mice, but whether this is related to preservation of immune function, and how it interacts with aging, is unclear. We compared phenotypic and functional characteristics of natural killer (NK) cells and T cells, across the lifespan, of calorie-restricted (CR) and control C57BL/6 and DBA/2 mice. Calorie restriction preserves a naïve T cell phenotype and an immature NK cell phenotype as mice age. The splenic T cell populations of CR mice had higher proportions of CD11a - CD44 lo cells, lower expression of TRAIL, KLRG1, and CXCR3, and higher expression of CD127, compared to control mice. Similarly, splenic NK cells from CR mice had higher proportions of less differentiated CD11b - CD27 + cells and correspondingly lower proportions of highly differentiated CD11b + CD27 - NK cells. Within each of these subsets, cells from CR mice had higher expression of CD127, CD25, TRAIL, NKG2A/C/E, and CXCR3 and lower expression of KLRG1 and Ly49 receptors compared to controls. The effects of calorie restriction on lymphoid cell populations in lung, liver, and lymph nodes were identical to those seen in the spleen, indicating that this is a system-wide effect. The impact of calorie restriction on NK cell and T cell maturation is much more profound than the effect of aging and, indeed, calorie restriction attenuates these age-associated changes. Importantly, the effects of calorie restriction on lymphocyte maturation were more marked in C57BL/6 than in DBA/2J mice indicating that delayed lymphocyte maturation correlates with extended lifespan. These findings have implications for understanding the interaction between nutritional status, immunity, and healthy lifespan in aging populations.

Regulation of exocytotic fusion by cell inflation.

PubMed Central

Solsona, C; Innocenti, B; Fernández, J M

1998-01-01

We have inflated patch-clamped mast cells by 3.8 +/- 1.6 times their volume by applying a hydrostatic pressure of 5-15 cm H2O to the interior of the patch pipette. Inflation did not cause changes in the cell membrane conductance and caused only a small reversible change in the cell membrane capacitance (36 +/- 5 fF/cm H2O). The specific cell membrane capacitance of inflated cells was found to be 0.5 microF/cm2. High-resolution capacitance recordings showed that inflation reduced the frequency of exocytotic fusion events by approximately 70-fold, with the remaining fusion events showing an unusual time course. Shortly after the pressure was returned to 0 cm H2O, mast cells regained their normal size and appearance and degranulated completely, even after remaining inflated for up to 60 min. We interpret these observations as an indication that inflated mast cells reversibly disassemble the structures that regulate exocytotic fusion. Upon returning to its normal size, the cell cytosol reassembles the fusion pore scaffolds and allows exocytosis to proceed, suggesting that exocytotic fusion does not require soluble proteins. Reassembly of the fusion pore can be prevented by inflating the cells with solutions containing the protease pronase, which completely blocked exocytosis. We also interpret these results as evidence that the activity of the fusion pore is sensitive to the tension of the plasma membrane. PMID:9533718

Cellular entry via an actin and clathrin-dependent route is required for Lv2 restriction of HIV-2.

PubMed

Harrison, I P; McKnight, A

2011-06-20

Lv2 is a human factor that restricts infection of some HIV-2 viruses after entry into particular target cells. HIV-2 MCR is highly susceptible to Lv2 whereas HIV-2 MCN is not. The block is after reverse transcription but prior to nuclear entry. The viral determinants for this restriction have been mapped to the HIV-2 envelope and the capsid genes. Our model of Lv2 restriction suggests that the route taken into a cell is important in determining whether a productive infection occurs. Here we characterised the infectious routes used by MCN and MCR using chemical compounds and molecular techniques to distinguish between potential pathways. Our results suggest that susceptible MCR can enter restrictive HeLa(CD4) cells via two pathways; a clathrin/AP2 mediated endocytic route that is sensitive to Lv2 restriction and an alternative, non-clathrin mediated route, which results in more efficient infection. Copyright © 2011 Elsevier Inc. All rights reserved.

Zinc improves learning and memory abilities of fetal growth restriction rats and promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 axis activity.

PubMed

Zong, Lu; Wei, Xiaohua; Gou, Wenli; Huang, Pu; Lv, Ye

2017-12-29

Fetal growth restriction (FGR) is a well-known risk factor for cognitive dysfunction, especially for learning and memory abilities. However, knowledge about prevention and treatment methods of learning and memory abilities of fetal are limit. Here, Morris water maze and passive avoidance tests showed zinc supplementation could protect the impairment of the learning and memory abilities caused by FGR. As accumulating evidence suggested that insufficiency of placental trophoblast cell invasion was closely related to FGR fetal neurodevelopmental dysplasia, we further explored the relationship between zinc supplementation during pregnancy and placental trophoblast. Microarray identified 346 differently expressed genes in placental tissues with and without zinc supplementation, and GO and KEGG analyses showed these differently expressed genes were highly enriched in cell invasion and migration and STAT3 pathway. Protein-protein interaction(PPI) analysis found that STAT3 interacted with matrix metalloproteinase-2/9 (MMP-2/9). In vivo , western blot results authenticated that the expression levels of phospho-STAT3, STAT3, MMP-2 and MMP-9 were up-regulated in placental tissues after zinc treatment. To validate whether zinc could promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 activity. In vitro , Transwell assay was performed, and we observed that abilities of invasion and migration were obviously increased in zinc treated trophoblast cells. And phospho-STAT3, STAT3, MMP-2 and MMP-9 expression levels were correspondingly increased in zinc treated trophoblast cells, which were dose-dependent. Moreover, gain-of-function and loss-of-function of STAT3 confirmed that zinc promotes cell invasion and migration via regulating STAT3 mediated up-regulation of MMP-2/9 activity. We propose that activation of MMP-2/9 mediated by STAT3 may contribute to invasion and migration of trophoblast cells, which improved neurodevelopmental impairment of FGR rats probably via contributing to placental development. Our findings are the first to show a possible mechanism of reversing neurodevelopmental impairment of FGR rats by zinc supplementation, holding promise for the development of novel therapeutic modalities for learning and memory abilities impairment caused by FGR.

Influence of human cytomegalovirus infection on the NK cell receptor repertoire in children.

PubMed

Monsiváis-Urenda, Adriana; Noyola-Cherpitel, Daniel; Hernández-Salinas, Alba; García-Sepúlveda, Christian; Romo, Neus; Baranda, Lourdes; López-Botet, Miguel; González-Amaro, Roberto

2010-05-01

Human cytomegalovirus (hCMV) infection is usually asymptomatic but may cause disease in immunocompromised hosts. It has been reported that hCMV infection may shape the NK cell receptor (NKR) repertoire in adult individuals, promoting a variable expansion of the CD94/NKG2C+ NK cell subset. We explored the possible relationship between this viral infection and the expression pattern of different NKR including CD94/NKG2C, CD94/NKG2A, immunoglobulin-like transcript 2 (ILT2, CD85j), KIR2DL1/2DS1, KIR3DL1, and CD161 in peripheral blood lymphocytes from healthy children, seropositive (n=21) and seronegative (n=20) for hCMV. Consistent with previous observations in adults, a positive serology for hCMV was associated with increased numbers of NKG2C+ NK and T cells as well as with ILT2+ T lymphocytes. Moreover, the proportions of CD161+ and NKG2C+CD56-CD3- NK cells also tended to be increased in hCMV+ individuals. Excretion of the virus was associated with higher proportions of NKG2C+ NK cells. Altogether, these data reveal that hCMV may have a profound influence on the NKR repertoire in early childhood.

Influence of enamel/dentin thickness on the toxic and esthetic effects of experimental in-office bleaching protocols.

PubMed

de Oliveira Duque, C C; Soares, D G; Basso, F G; Hebling, J; de Souza Costa, C A

2017-11-01

This paper aims to assess the whitening effectiveness and toxicity of tooth-bleaching protocols applied to enamel/dentin disks simulating mandibular incisors (ICs) and premolars (PMs). A 10% hydrogen peroxide (H 2 O 2 ) gel was applied for 3 × 15, 1 × 15, or 1 × 5 min to enamel/dentin disks simulating mandibular ICs and PMs, and the trans-enamel and trans-dentinal diffusion products were applied to human dental pulp cells (1 h). Professional therapy (35% H 2 O 2 -3 × 15 min) was used as positive control, and non-bleached samples were used as negative control. Cell viability and morphology, oxidative stress generation, and odontoblastic marker expression were assessed. The H 2 O 2 diffusion and enamel color change (ΔE) were also analyzed. The 10% H 2 O 2 gel induced significant cell viability reduction only when applied 3 × 15 min, with the intensity of oxidative stress and down-regulation of odontoblastic markers being higher in the IC group. The other experimental bleaching protocols caused slight alterations regarding the cell parameters evaluated, with intensity being related to enamel/dentin thickness. These effects were also correlated with higher H 2 O 2 diffusion in the IC group. ΔE values similar as positive control were found for the 10% 3 × 15 and 1 × 15 protocols on IC group, after 4 and 6 sessions. Application of a 10% H 2 O 2 bleaching gel for 15 or 45 min to thin dental substrate significantly minimizes cell toxicity in comparison with highly concentrated gels associated with similar esthetic outcomes by increasing the number of bleaching sessions. Bleaching gels with 10% H 2 O 2 applied in small teeth for short periods may be an interesting alternative to obtain whitening effectiveness without causing toxicity to pulp cells, which may be able to reduce the tooth hypersensitivity claimed by patients.

Inhibition of antibody synthesis by histamine in concanavalin A-treated mice: the possible role of glucocorticosteroids.

PubMed

Badger, A M; Griswold, D E; DiMartino, M J; Poste, G

1982-09-01

Administration of histamine (50 mg/kg) to BALB/C mice injected with concanavalin A (Con A) (100 micrograms, i.v.) 24 hr previously, results in a marked decrease in antibody synthesis to sheep red blood cells (SRBC) injected 2 hr later. This phenomenon occurs with nonimmunosuppressive doses of Con A and is strain-specific. It does not take place in the response to the T-independent antigen polyvinylpyrrolidone (PVP) or if histamine is administered after the antigen. Adoptive transfer of normal syngeneic cells at the same time as antigen does not reverse this effect. Excess suppressor cell generation was excluded by co-cultivation of treated spleen cells with normal cells in vitro and by determining their antibody response to SRBC 5 days later. 2-Methylhistamine, a histamine type 1 (H1) receptor agonist, mimicks the effect of histamine whereas dimaprit, a histamine type 2 (H2) receptor agonist, does not. Because histamine interaction with H1 receptors causes the release of adrenocorticotropic hormone (ACTH), we examined the effects of ACTH and corticosterone in this system and found that both could mimick the effect of histamine. These results suggest that the interaction of histamine with H1 receptors causes the release of glucocorticosteroids that may interfere with either Con A-activated T helper cell function or macrophage processing of T-dependent antigen.

The natural compound Guttiferone F sensitizes prostate cancer to starvation induced apoptosis via calcium and JNK elevation.

PubMed

Li, Xin; Lao, Yuanzhi; Zhang, Hong; Wang, Xiaoyu; Tan, Hongsheng; Lin, Zhixiu; Xu, Hongxi

2015-04-11

In a cytotoxicity screen in serum-free medium, Guttiferone F showed strong growth inhibitory effect against prostate cancer cells. Prostate cancer cells LNCaP and PC3 were treated with Guttiferone F in serum depleted medium. Sub-G1 phase distributions were estimated with flow cytometry. Mitochondrial disruption was observed under confocal microscope using Mitotracker Red staining. Gene and protein expression changes were detected by real-time PCR and Western blotting. Ca(2+) elevation was examined by Fluo-4 staining under fluorescence microscope. PC3 xenografts in mice were examined by immunohistochemical analysis. Guttiferone F had strong growth inhibitory effect against prostate cancer cell lines under serum starvation. It induced a significant increase in sub-G1 fraction and DNA fragmentation. In serum-free medium, Guttiferone F triggered mitochondria dependent apoptosis by regulating Bcl-2 family proteins. In addition, Guttiferone F attenuated the androgen receptor expression and phosphorylation of ERK1/2, while activating the phosphorylation of JNK and Ca(2+) flux. Combination of caloric restriction with Guttiferone F in vivo could increase the antitumor effect without causing toxicity. Guttiferone F induced prostate cancer cell apoptosis under serum starvation via Ca(2+) elevation and JNK activation. Combined with caloric restriction, Guttiferone F exerted significant growth inhibition of PC3 cells xenograft in vivo. Guttiferone F is therefore a potential anti-cancer compound.

Alterations in adenosine triphosphate and energy charge in cultured endothelial and P388D1 cells after oxidant injury.

PubMed Central

Spragg, R G; Hinshaw, D B; Hyslop, P A; Schraufstätter, I U; Cochrane, C G

1985-01-01

To investigate mechanisms whereby oxidant injury of cells results in cell dysfunction and death, cultured endothelial cells or P388D1 murine macrophage-like cells were exposed to oxidants including H2O2, O2-. (generated by the enzymatic oxidation of xanthine), or to stimulated polymorphonuclear leukocytes (PMN). Although Trypan Blue exclusion was not diminished before 30 min, cellular ATP was found to fall to less than 30% of control values within 3 min of exposure to 5 mM H2O2. Stimulated PMN plus P388D1 caused a 50% fall in cellular ATP levels. During the first minutes of oxidant injury, total adenylate content of cells fell by 85%. Cellular ADP increased 170%, AMP increased 900%, and an 83% loss of ATP was accompanied by a stoichiometric increase in IMP and inosine. Calculated energy charge [(ATP + 1/2 AMP)/(ATP + ADP + AMP)] fell from 0.95 to 0.66. Exposure of P388D1 to oligomycin plus 2-deoxyglucose (which inhibit oxidative and glycolytic generation of ATP, respectively) resulted in a rate of ATP fall similar to that induced by H2O2. In addition, nucleotide alterations induced by exposure to oligomycin plus 2-deoxyglucose were qualitatively similar to those induced by the oxidant. Loss of cell adenylates could not be explained by arrest of de novo purine synthesis or increased ATP consumption by the Na+-K+ ATPase or the mitochondrial F0-ATPase. These results indicate that H2O2 causes a rapid and profound fall in cellular ATP levels similar to that seen when ATP production is arrested by metabolic inhibitors. PMID:2997279

Effects of recovery sleep after one work week of mild sleep restriction on interleukin-6 and cortisol secretion and daytime sleepiness and performance.

PubMed

Pejovic, Slobodanka; Basta, Maria; Vgontzas, Alexandros N; Kritikou, Ilia; Shaffer, Michele L; Tsaoussoglou, Marina; Stiffler, David; Stefanakis, Zacharias; Bixler, Edward O; Chrousos, George P

2013-10-01

One workweek of mild sleep restriction adversely impacts sleepiness, performance, and proinflammatory cytokines. Many individuals try to overcome these adverse effects by extending their sleep on weekends. To assess whether extended recovery sleep reverses the effects of mild sleep restriction on sleepiness/alertness, inflammation, and stress hormones, 30 healthy young men and women (mean age ± SD, 24.7 ± 3.5 yr; mean body mass index ± SD, 23.6 ± 2.4 kg/m(2)) participated in a sleep laboratory experiment of 13 nights [4 baseline nights (8 h/night), followed by 6 sleep restriction nights (6 h/night) and 3 recovery nights (10 h/night)]. Twenty-four-hour profiles of circulating IL-6 and cortisol, objective and subjective daytime sleepiness (Multiple Sleep Latency Test and Stanford Sleepiness Scale), and performance (Psychomotor Vigilance Task) were assessed on days 4 (baseline), 10 (after 1 wk of sleep restriction), and 13 (after 2 nights of recovery sleep). Serial 24-h IL-6 plasma levels increased significantly during sleep restriction and returned to baseline after recovery sleep. Serial 24-h cortisol levels during restriction did not change compared with baseline, but after recovery they were significantly lower. Subjective and objective sleepiness increased significantly after restriction and returned to baseline after recovery. In contrast, performance deteriorated significantly after restriction and did not improve after recovery. Extended recovery sleep over the weekend reverses the impact of one work week of mild sleep restriction on daytime sleepiness, fatigue, and IL-6 levels, reduces cortisol levels, but does not correct performance deficits. The long-term effects of a repeated sleep restriction/sleep recovery weekly cycle in humans remain unknown.

Effects of recovery sleep after one work week of mild sleep restriction on interleukin-6 and cortisol secretion and daytime sleepiness and performance

PubMed Central

Pejovic, Slobodanka; Basta, Maria; Kritikou, Ilia; Shaffer, Michele L.; Tsaoussoglou, Marina; Stiffler, David; Stefanakis, Zacharias; Bixler, Edward O.; Chrousos, George P.

2013-01-01

One workweek of mild sleep restriction adversely impacts sleepiness, performance, and proinflammatory cytokines. Many individuals try to overcome these adverse effects by extending their sleep on weekends. To assess whether extended recovery sleep reverses the effects of mild sleep restriction on sleepiness/alertness, inflammation, and stress hormones, 30 healthy young men and women (mean age ± SD, 24.7 ± 3.5 yr; mean body mass index ± SD, 23.6 ± 2.4 kg/m2) participated in a sleep laboratory experiment of 13 nights [4 baseline nights (8 h/night), followed by 6 sleep restriction nights (6 h/night) and 3 recovery nights (10 h/night)]. Twenty-four-hour profiles of circulating IL-6 and cortisol, objective and subjective daytime sleepiness (Multiple Sleep Latency Test and Stanford Sleepiness Scale), and performance (Psychomotor Vigilance Task) were assessed on days 4 (baseline), 10 (after 1 wk of sleep restriction), and 13 (after 2 nights of recovery sleep). Serial 24-h IL-6 plasma levels increased significantly during sleep restriction and returned to baseline after recovery sleep. Serial 24-h cortisol levels during restriction did not change compared with baseline, but after recovery they were significantly lower. Subjective and objective sleepiness increased significantly after restriction and returned to baseline after recovery. In contrast, performance deteriorated significantly after restriction and did not improve after recovery. Extended recovery sleep over the weekend reverses the impact of one work week of mild sleep restriction on daytime sleepiness, fatigue, and IL-6 levels, reduces cortisol levels, but does not correct performance deficits. The long-term effects of a repeated sleep restriction/sleep recovery weekly cycle in humans remain unknown. PMID:23941878

The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells.

PubMed

Azijli, Kaamar; van Roosmalen, Ingrid A M; Smit, Jorn; Pillai, Saravanan; Fukushima, Masakazu; de Jong, Steven; Peters, Godefridus J; Bijnsdorp, Irene V; Kruyt, Frank A E

2014-06-01

TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL. Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms. The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression. TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.

Abnormal Pulmonary Function in Adults with Sickle Cell Anemia

PubMed Central

Klings, Elizabeth S.; Wyszynski, Diego F.; Nolan, Vikki G.; Steinberg, Martin H.

2006-01-01

Rationale: Pulmonary complications of sickle cell anemia (Hb-SS) commonly cause morbidity, yet few large studies of pulmonary function tests (PFTs) in this population have been reported. Objectives: PFTs (spirometry, lung volumes, and diffusion capacity for carbon monoxide [DLCO]) from 310 adults with Hb-SS were analyzed to determine the pattern of pulmonary dysfunction and their association with other systemic complications of sickle cell disease. Methods: Raw PFT data were compared with predicted values. Each subject was subclassified into one of five groups: obstructive physiology, restrictive physiology, mixed obstructive/restrictive physiology, isolated low DLCO, or normal. The association between laboratory data of patients with decreased DLCO or restrictive physiology and those of normal subjects was assessed by multivariate linear regression. Measurements and Main Results: Normal PFTs were present in only 31 of 310 (10%) patients. Overall, adults with Hb-SS were characterized by decreased total lung capacities (70.2 ± 14.7% predicted) and DlCO (64.5 ± 19.9%). The most common PFT patterns were restrictive physiology (74%) and isolated low DlCO (13%). Decreased DLCO was associated with thrombocytosis (p = 0.05), with hepatic dysfunction (elevated alanine aminotransferase; p = 0.07), and a trend toward renal dysfunction (elevated blood urea nitrogen and creatinine; p = 0.05 and 0.07, respectively). Conclusions: Pulmonary function is abnormal in 90% of adult patients with Hb-SS. Common abnormalities include restrictive physiology and decreased DLCO. Decreased DLCO may indicate more severe sickle vasculopathy characterized by impaired hepatic and renal function. PMID:16556694

Acidic pH promotes intervertebral disc degeneration: Acid-sensing ion channel -3 as a potential therapeutic target.

PubMed

Gilbert, Hamish T J; Hodson, Nathan; Baird, Pauline; Richardson, Stephen M; Hoyland, Judith A

2016-11-17

The aetiology of intervertebral disc (IVD) degeneration remains poorly understood. Painful IVD degeneration is associated with an acidic intradiscal pH but the response of NP cells to this aberrant microenvironmental factor remains to be fully characterised. The aim here was to address the hypothesis that acidic pH, similar to that found in degenerate IVDs, leads to the altered cell/functional phenotype observed during IVD degeneration, and to investigate the involvement of acid-sensing ion channel (ASIC) -3 in the response. Human NP cells were treated with a range of pH, from that of a non-degenerate (pH 7.4 and 7.1) through to mildly degenerate (pH 6.8) and severely degenerate IVD (pH 6.5 and 6.2). Increasing acidity of pH caused a decrease in cell proliferation and viability, a shift towards matrix catabolism and increased expression of proinflammatory cytokines and pain-related factors. Acidic pH resulted in an increase in ASIC-3 expression. Importantly, inhibition of ASIC-3 prevented the acidic pH induced proinflammatory and pain-related phenotype in NP cells. Acidic pH causes a catabolic and degenerate phenotype in NP cells which is inhibited by blocking ASIC-3 activity, suggesting that this may be a useful therapeutic target for treatment of IVD degeneration.

Activation of Nrf2 is required for up-regulation of the π class of glutathione S-transferase in rat primary hepatocytes with L-methionine starvation.

PubMed

Lin, Ai-Hsuan; Chen, Haw-Wen; Liu, Cheng-Tze; Tsai, Chia-Wen; Lii, Chong-Kuei

2012-07-04

Numerous genes expression is regulated in response to amino acid shortage, which helps organisms adapt to amino acid limitation. The expression of the π class of glutathione (GSH) S-transferase (GSTP), a highly inducible phase II detoxification enzyme, is regulated mainly by activates activating protein 1 (AP-1) binding to the enhancer I of GSTP (GPEI). Here we show the critical role of nuclear factor erythroid-2-related factor 2 (Nrf2) in up-regulating GSTP gene transcription. Primary rat hepatocytes were cultured in a methionine-restricted medium, and immunoblotting and RT-PCR analyses showed that methionine restriction time-dependently increased GSTP protein and mRNA expression over a 48 h period. Nrf2 translocation to the nucleus, nuclear proteins binding to GPEI, and antioxidant response element (ARE) luciferase reporter activity were increased by methionine restriction as well as by l-buthionine sulfoximine (BSO), a GSH synthesis inhibitor. Transfection with Nrf2 siRNA knocked down Nrf2 expression and reversed the methionine-induced GSTP expression and GPEI binding activity. Chromatin immunoprecipitation assay confirmed the binding of Nrf2 to the GPEI. Phosphorylation of extracellular signal-regulated kinase 2 (ERK2) was increased in methionine-restricted and BSO-treated cells. ERK2 siRNA abolished methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression. Our results suggest that the up-regulation of GSTP gene transcription in response to methionine restriction likely occurs via the ERK-Nrf2-GPEI signaling pathway.

Can apple antioxidants inhibit tumor cell proliferation? Generation of H(2)O(2) during interaction of phenolic compounds with cell culture media.

PubMed

Lapidot, Tair; Walker, Michael D; Kanner, Joseph

2002-05-22

It has recently been suggested that the ability of apple extracts to inhibit proliferation of tumor cells in vitro may be due to phenolic/flavonoid antioxidants. Our study demonstrates that this inhibition is caused indirectly by H(2)O(2) generated through interaction of the phenolics with the cell culture media. The results indicate that many previously reported effects of flavonoids and phenolic compounds on cultured cells may result from similar artifactual generation of oxidative stress. We suggest that in order to prevent such artifacts, the use of catalase and/or metmyoglobin in the presence of reducing agents should be considered as a method to decompose H(2)O(2) and prevent generation of other reactive oxygen species, which could affect cell proliferation. The use of tumor cells and "nontumor cells" in a bioassay to measure antioxidant activity, in this context, is potentially misleading and should be applied with caution.

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells.

PubMed

Jiao, Jiao; Sun, Ling; Zhou, Benguo; Gao, Zhengliang; Hao, Yu; Zhu, Xiaoping; Liang, Yuancun

2014-08-15

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H2O2 accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

PubMed

Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

2017-11-01

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.

Differential effect of grape seed extract against human non-small-cell lung cancer cells: the role of reactive oxygen species and apoptosis induction.

PubMed

Tyagi, Alpna; Raina, Komal; Gangar, Subhash; Kaur, Manjinder; Agarwal, Rajesh; Agarwal, Chapla

2013-01-01

The present study examines grape seed extract (GSE) efficacy against a series of non-small-cell lung cancer (NSCLC) cell lines that differ in their Kras and p53 status to establish GSE potential as a cytotoxic agent against a wide range of lung cancer cells. GSE suppressed growth and induced apoptotic death in NSCLC cells irrespective of their k-Ras status, with more sensitivity toward H460 and H322 (wt k-Ras) than A549 and H1299 cells (mutated k-Ras). Mechanistic studies in A549 and H460 cells, selected, based on comparative efficacy of GSE at higher and lower doses, respectively, showed that apoptotic death involves cytochrome c release associated caspases 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, strong phosphorylation of ERK1/2 and JNK1/2, downregulation of cell survival proteins, and upregulated proapoptotic Bak expression. Importantly, GSE treatment caused a strong superoxide radical-associated oxidative stress, significantly decreased intracellular reduced glutathione levels, suggesting, for the first time, the involvement of GSE-caused oxidative stress in its apoptotic inducing activity in these cells. Because GSE is a widely-consumed dietary agent with no known untoward effects, our results support future studies to establish GSE efficacy and usefulness against NSCLC control.

Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

PubMed

Saggese, Taryn; Thambyah, Ashvin; Wade, Kelly; McGlashan, Susan Read

2018-05-01

Objective The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. Design Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. Results Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. Conclusion This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis.

PubMed

Walia, V; Yu, Y; Cao, D; Sun, M; McLean, J R; Hollier, B G; Cheng, J; Mani, S A; Rao, K; Premkumar, L; Elble, R C

2012-04-26

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.

Loss of breast epithelial marker hCLCA2 promotes epithelial to mesenchymal transition and indicates higher risk of metastasis

PubMed Central

Walia, Vijay; Yu, Yang; Cao, Deshou; Sun, Miao; McLean, Janel R.; Hollier, Brett G.; Cheng, Jiming; Mani, Sendurai A.; Rao, Krishna; Premkumar, Louis; Elble, Randolph

2013-01-01

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of EMT by cell dilution, TGFbeta, or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral shRNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18 year period among breast cancer patients compared to lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells. PMID:21909135

Six and 12 Weeks of Caloric Restriction Increases β Cell Function and Lowers Fasting and Postprandial Glucose Concentrations in People with Type 2 Diabetes123

PubMed Central

Sathananthan, Matheni; Shah, Meera; Edens, Kim L; Grothe, Karen B; Piccinini, Francesca; Farrugia, Luca P; Micheletto, Francesco; Man, Chiara Dalla; Cobelli, Claudio; Rizza, Robert A; Camilleri, Michael; Vella, Adrian

2015-01-01

Background: Caloric restriction alone has been shown to improve insulin action and fasting glucose metabolism; however, the mechanism by which this occurs remains uncertain. Objective: We sought to quantify the effect of caloric restriction on β cell function and glucose metabolism in people with type 2 diabetes. Methods: Nine subjects (2 men, 7 women) with type 2 diabetes [BMI (in kg/m2): 40.6 ± 1.4; age: 58 ± 3 y; glycated hemoglobin: 6.9% ± 0.2%] were studied using a triple-tracer mixed meal after withdrawal of oral diabetes therapy. The oral minimal model was used to measure β cell function. Caloric restriction limited subjects to a pureed diet (<900 kcal/d) for the 12 wk of study. The studies were repeated after 6 and 12 wk of caloric restriction. Results: Fasting glucose concentrations decreased significantly from baseline after 6 wk of caloric restriction with no further reduction after a further 6 wk of caloric restriction (9.8 ± 1.3, 5.9 ± 0.2, and 6.2 ± 0.3 mmol/L at baseline and after 6 and 12 wk of caloric restriction, respectively; P = 0.01) because of decreased fasting endogenous glucose production (EGP: 20.4 ± 1.1, 16.2 ± 0.8, and 17.4 ± 1.1 μmol · kg−1 · min−1 at baseline and after 6 and 12 wk of caloric restriction, respectively; P = 0.03). These changes were accompanied by an improvement in β cell function measured by the disposition index (189 ± 51, 436 ± 68, and 449 ± 67 10−14 dL · kg−1 · min−2 · pmol−1 at baseline and after 6 and 12 wk of caloric restriction, respectively; P = 0.01). Conclusions: Six weeks of caloric restriction lowers fasting glucose and EGP with accompanying improvements in β cell function in people with type 2 diabetes. An additional 6 wk of caloric restriction maintained the improvement in glucose metabolism. This trial was registered at clinicaltrials.gov as NCT01094054. PMID:26246321

Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8+ T lymphocytes

PubMed Central

Singh, Shailbala; Toro, Haroldo; Tang, De-Chu; Briles, Worthie E.; Yates, Linda M.; Kopulos, Renee T.; Collisson, Ellen W.

2010-01-01

Avian influenza virus (AIV) specific CD8+ T lymphocyte responses stimulated by intramuscular administration of an adenovirus (Ad) vector expressing either HA or NP were evaluated in chickens following ex vivo stimulation by non-professional antigen presenting cells. The CD8+ T lymphocyte responses were AIV specific, MHC-I restricted, and cross-reacted with heterologousH7N2 AIV strain. Specific effector responses, at 10 days post-inoculation (p.i.), were undetectable at 2 weeks p.i., and memory responses were detected from 3 to 8 weeks p.i. Effector memory responses, detected 1 week following a booster inoculation, were significantly greater than the primary responses and, within 7 days, declined to undetectable levels. Inoculation of an Ad-vector expressing human NP resulted in significantly greater MHC restricted, activation of CD8+ T cell responses specific for AIV. Decreases in all responses with time were most dramatic with maximum activation of T cells as observed following effector and effector memory responses. PMID:20557918

H{sub 2}S does not regulate proliferation via T-type Ca{sup 2+} channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elies, Jacobo; Johnson, Emily; Boyle, John P.

T-type Ca{sup 2+} channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca{sup 2+} channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca{sup 2+} channel Cav3.2 is selectively inhibited by hydrogen sulfide (H{sub 2}S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H{sub 2}S could account for the anti-proliferative effects of this gasotransmitter. H{sub 2}S suppressed proliferation inmore » HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H{sub 2}S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H{sub 2}S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca{sup 2+} channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H{sub 2}S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca{sup 2+} channel isoform was the H{sub 2}S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca{sup 2+} channel-mediated proliferation by H{sub 2}S is independent of the channels’ sensitivity to H{sub 2}S. - Highlights: • T-type Ca{sup 2+} channels regulate proliferation and are sensitive to the gasotransmitters CO and H{sub 2}S. • H{sub 2}S reduced proliferation in HEK293 cells expressing the H{sub 2}S sensitive Cav3.2 channel. • H{sub 2}S also inhibited proliferation in non-transfected cells and HEK293 cells expressing Cav3.1. • Native smooth muscle cells primarily express Cav3.1. Their proliferation was also inhibited by H{sub 2}S. • Unlike CO, H{sub 2}S does not regulate smooth muscle proliferation via T-type Ca{sup 2+} channel inhibition.« less

Biological functions of hCG and hCG-related molecules

PubMed Central

2010-01-01

Background hCG is a term referring to 4 independent molecules, each produced by separate cells and each having completely separate functions. These are hCG produced by villous syncytiotrophoblast cells, hyperglycosylated hCG produced by cytotrophoblast cells, free beta-subunit made by multiple primary non-trophoblastic malignancies, and pituitary hCG made by the gonadotrope cells of the anterior pituitary. Results and discussion hCG has numerous functions. hCG promotes progesterone production by corpus luteal cells; promotes angiogenesis in uterine vasculature; promoted the fusion of cytotrophoblast cell and differentiation to make syncytiotrophoblast cells; causes the blockage of any immune or macrophage action by mother on foreign invading placental cells; causes uterine growth parallel to fetal growth; suppresses any myometrial contractions during the course of pregnancy; causes growth and differentiation of the umbilical cord; signals the endometrium about forthcoming implantation; acts on receptor in mother's brain causing hyperemesis gravidarum, and seemingly promotes growth of fetal organs during pregnancy. Hyperglycosylated hCG functions to promote growth of cytotrophoblast cells and invasion by these cells, as occurs in implantation of pregnancy, and growth and invasion by choriocarcinoma cells. hCG free beta-subunit is produced by numerous non-trophoblastic malignancies of different primaries. The detection of free beta-subunit in these malignancies is generally considered a sign of poor prognosis. The free beta-subunit blocks apoptosis in cancer cells and promotes the growth and malignancy of the cancer. Pituitary hCG is a sulfated variant of hCG produced at low levels during the menstrual cycle. Pituitary hCG seems to mimic luteinizing hormone actions during the menstrual cycle. PMID:20735820

Investigation of Laser-Induced Retinal Damage: Wavelength and Pulsewidth Dependent Mechanisms

DTIC Science & Technology

1994-06-30

Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1991;1072:129-57. 2. Artuc M, Ramshad M, Kappus H. Studies...M, Reinhold C, Kappus H. DNA damage caused by laser light activated hematoporphyrin derivatives in isolated nuclei of human melanoma cells. Arch

Glucose deprivation elicits phenotypic plasticity via ZEB1-mediated expression of NNMT

PubMed Central

Kanska, Justyna; Aspuria, Paul-Joseph P.; Taylor-Harding, Barbie; Spurka, Lindsay; Funari, Vincent; Orsulic, Sandra; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

2017-01-01

Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cells. We found that cells resistant to glucose starvation (glucose-restricted cells) demonstrated increased metabolic plasticity that was dependent on NNMT (Nicotinamide N-methyltransferase) expression. We further show that ZEB1 induced NNMT, rendered cells resistant to glucose deprivation and recapitulated metabolic adaptations and mesenchymal gene expression observed in glucose-restricted cells. NNMT depletion reversed metabolic plasticity in glucose-restricted cells and prevented de novo formation of glucose-restricted colonies. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. NNMT protein levels were also elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits mesenchymal gene expression, as well as phenotypic and metabolic plasticity in ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in high-grade serous ovarian cancer (HGSC). PMID:28412735

Work of breathing using different interfaces in spontaneous positive pressure ventilation: helmet, face-mask, and endotracheal tube.

PubMed

Oda, Shinya; Otaki, Kei; Yashima, Nozomi; Kurota, Misato; Matsushita, Sachiko; Kumasaka, Airi; Kurihara, Hutaba; Kawamae, Kaneyuki

2016-08-01

Noninvasive positive pressure ventilation (NPPV) using a helmet is expected to cause inspiratory trigger delay due to the large collapsible and compliant chamber. We compared the work of breathing (WOB) of NPPV using a helmet or a full face-mask with that of invasive ventilation by tracheal intubation. We used a lung model capable of simulating spontaneous breathing (LUNGOO; Air Water Inc., Japan). LUNGOO was set at compliance (C) = 50 mL/cmH2O and resistance (R) = 5 cmH2O/L/s for normal lung simulation, C = 20 mL/cmH2O and R = 5 cmH2O/L/s for restrictive lung, and C = 50 mL/cmH2O and R = 20 cmH2O/L/s for obstructive lung. Muscle pressure was fixed at 25 cmH2O and respiratory rate at 20 bpm. Pressure support ventilation and continuous positive airway pressure were performed with each interface placed on a dummy head made of reinforced plastic that was connected to LUNGOO. We tested the inspiratory WOB difference between the interfaces with various combinations of ventilator settings (positive end-expiratory pressure 5 cmH2O; pressure support 0, 5, and 10 cmH2O). In the normal lung and restrictive lung models, WOB decreased more with the face-mask than the helmet, especially when accompanied by the level of pressure support. In the obstructive lung model, WOB with the helmet decreased compared with the other two interfaces. In the mixed lung model, there were no significant differences in WOB between the three interfaces. NPPV using a helmet is more effective than the other interfaces for WOB in obstructive lung disease.

Kinetic analysis of dihydroxyacetone production from crude glycerol by immobilized cells of Gluconobacter oxydans MTCC 904.

PubMed

Dikshit, Pritam Kumar; Moholkar, Vijayanand S

2016-09-01

The present study has investigated kinetic features of bioconversion of biodiesel-derived crude glycerol to dihydroxyacetone with immobilized Gluconobacter oxydans cells using modified Haldane substrate-inhibition model. The results have been compared against free cells and pure glycerol. Relative variations in the kinetic parameters KS, KI, Vmax, n and X reveal that immobilized G. oxydans cells (on PU foam substrate) with crude glycerol as substrate give higher order of inhibition (n) and lower maximum reaction velocities (Vmax). These results are essentially implications of substrate transport restrictions across immobilization matrix, which causes retention of substrate in the matrix and reduction in fractional available substrate (X) for the cells. This causes reduction in both KS (substrate concentration at Vmax/2) and KI (inhibition constant) as compared to free cells. For immobilized cells, substrate concentration (Smax) corresponding to Vmax is practically same for both pure and crude glycerol as substrate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells

PubMed Central

Zhu, Shu; Ding, Siyuan; Wang, Penghua; Wei, Zheng; Pan, Wen; Palm, Noah W; Yang, Yi; Yu, Hua; Li, Hua-Bing; Wang, Geng; Lei, Xuqiu; de Zoete, Marcel R.; Zhao, Jun; Zheng, Yunjiang; Chen, Haiwei; Zhao, Yujiao; Jurado, Kellie A.; Feng, Ningguo; Shan, Liang; Kluger, Yuval; Lu, Jun; Abraham, Clara; Fikrig, Erol; Greenberg, Harry B.; Flavell, Richard A.

2018-01-01

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide1. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling2–5, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens. PMID:28636595

The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells

PubMed Central

Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M.; Ballana, Ester

2016-01-01

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004

Down-regulation of poison ivy/oak-induced contact sensitivity by treatment with a class II MHC binding peptide:hapten conjugate.

PubMed

Gelber, C; Gemmell, L; McAteer, D; Homola, M; Swain, P; Liu, A; Wilson, K J; Gefter, M

1997-03-01

Immune regulation of contact sensitivity to the poison ivy/oak catechol was studied at the level of class II MHC-restricted T cell recognition of hapten:peptide conjugates. In this study we have shown that 1) T cells from C3H/HeN (H-2k) mice, immunized with a synthetic I-Ak binding peptide coupled to 3-pentadecyl-catechol (PDC; a representative catechol in urushiol), recognized peptides derived from syngeneic cells linked to the same catechol; 2) T cells from draining lymph nodes of C3H/HeN mice skin-painted with PDC proliferated in response to a peptide carrier:PDC conjugate only when it was linked at the 7th, but not the 4th or the 10th, position on the peptide carrier; and 3) tolerization studies confirmed down-regulation of PDC-induced delayed-type hypersensitivity following treatment with a single I-Ak binding peptide carrying PDC covalently bound to a lysine residue at the middle (7th) TCR contact position. Tolerization with peptide:PDC conjugate resulted in abrogation of hapten-specific T cell proliferative responses that correlated with diminished IL-2 secretion. On the basis of these data we propose that it may be sufficient to couple the hapten at a single, well-chosen position on a carrier peptide to target a relevant population of T cells involved in contact sensitivity.

Hydrogen gas alleviates oxygen toxicity by reducing hydroxyl radical levels in PC12 cells

PubMed Central

Yu, Junchao; Yu, Qiuhong; Liu, Yaling; Zhang, Ruiyun; Xue, Lianbi

2017-01-01

Hyperbaric oxygen (HBO) therapy through breathing oxygen at the pressure of above 1 atmosphere absolute (ATA) is useful for varieties of clinical conditions, especially hypoxic-ischemic diseases. Because of generation of reactive oxygen species (ROS), breathing oxygen gas at high pressures can cause oxygen toxicity in the central nervous system, leading to multiple neurological dysfunction, which limits the use of HBO therapy. Studies have shown that Hydrogen gas (H2) can diminish oxidative stress and effectively reduce active ROS associated with diseases. However, the effect of H2 on ROS generated from HBO therapy remains unclear. In this study, we investigated the effect of H2 on ROS during HBO therapy using PC12 cells. PC12 cells cultured in medium were exposed to oxygen gas or mixed oxygen gas and H2 at 1 ATA or 5 ATA. Cells viability and oxidation products and ROS were determined. The data showed that H2 promoted the cell viability and inhibited the damage in the cell and mitochondria membrane, reduced the levels of lipid peroxidation and DNA oxidation, and selectively decreased the levels of •OH but not disturbing the levels of O2•-, H2O2, or NO• in PC12 cells during HBO therapy. These results indicated that H2 effectively reduced •OH, protected cells against oxygen toxicity resulting from HBO therapy, and had no effect on other ROS. Our data supported that H2 could be potentially used as an antioxidant during HBO therapy. PMID:28362819

Combusted but not smokeless tobacco products cause DNA damage in oral cavity cells.

PubMed

Gao, Hong; Prasad, G L; Zacharias, Wolfgang

2014-05-01

The aim of this work was to investigate genomic DNA damage in human oral cavity cells after exposure to different tobacco product preparations (TPPs). The oral carcinoma cell line 101A, gingival epithelial cells HGEC, and gingival fibroblasts HGF were exposed to TPM (total particulate matter from 3R4F cigarettes), ST/CAS (2S3 smokeless tobacco extract in complete artificial saliva), and NIC (nicotine). Treatments were for 24 h using TPM at its EC-50 doses, ST/CAS and NIC at doses with equi-nicotine units, and high doses for ST/CAS and NIC. Comet assays showed that TPM, but not ST/CAS or NIC, caused substantial DNA breaks in cells; only the high ST/CAS dose caused weak DNA damage. These results were confirmed by immunofluorescence for γ-H2AX protein. These data revealed that the combusted TPP caused substantial DNA damage in all cell types, whereas the two non-combusted TPPs exerted no or only minimal DNA damage. They support epidemiologic evidence on the relative risk associated with consumption of non-combusted versus combusted tobacco products, and help to understand potential genotoxic effects of such products on oral cavity cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Influence of hydrogen-occluding-silica on migration and apoptosis in human esophageal cells in vitro.

PubMed

Li, Qiang; Tanaka, Yoshiharu; Miwa, Nobuhiko

2017-01-01

In the last decade, many studies have shown that hydrogen gas or hydrogen water can reduce the levels of reactive oxygen species in the living body. Molecular hydrogen has antioxidant and antiapoptotic effects and a preventive effect on oxidative stress-induced cell death. In the present study, we investigated solidified hydrogen-occluding-silica (H 2 -silica) that can release molecular hydrogen into cell culture medium because the use of hydrogen gas has strict handling limitations in hospital and medical facilities and laboratories, owing to its physicochemical characteristics. Human esophageal squamous cell carcinoma (KYSE-70) cells and normal human esophageal epithelial cells (HEEpiCs) were used to investigate the effects of H 2 -silica on cell viability and proliferation. Cell migration was examined with wound healing and culture-insert migration assays. The intracellular levels of reactive oxygen species were evaluated with a nitroblue tetrazolium assay. To assess the apoptotic status of the cells, the Bax/Bcl-2 ratio and cleaved caspase-3 were analyzed by western blot. The results showed that KYSE-70 cells and HEEpiCs were generally inhibited by H 2 -silica administration, and there was a significant proliferation-inhibitory effect in an H 2 -silica concentration-dependent manner compared with the control group ( P < 0.05) in KYSE-70. Apoptosis-inducing effect on KYSE-70 cells was observed in 10, 300, 600, and 1,200 ppm H 2 -silica, and only 1,200 ppm H 2 -silica caused a 2.4-fold increase in apoptosis in HEEpiCs compared with the control group as the index of Bax/Bcl-2. H 2 silica inhibited cell migration in KYSE-70 cells, and high concentrations had a cytotoxic effect on normal cells. These findings should provide insights into the mechanism of inhibition of H 2 -silica on human cancer cells in vitro .

Hydrogen peroxide inhibits iodide influx and enhances iodide efflux in cultured FRTL-5 rat thyroid cells.

PubMed

Sugawara, M; Yamaguchi, D T; Lee, H Y; Yanagisawa, K; Murakami, S; Summer, C N; Johnson, D G; Levin, S R

1990-05-01

This study describes the effects of hydrogen peroxide on the two iodide transport systems, I influx and I efflux, in the cultured FRTL-5 rat thyroid cells. I influx was measured by the amount of I taken up by the cells during incubation with Na125I and NaI for 7 min, and I efflux was measured by calculating the rate of 125I release from the 125I-loaded cells in the presence and absence of 5 mmol/l H2O2. Exposure to greater than 100 mumol/l H2O2 for 40 min caused a significant inhibition of I influx; the inhibition was reversible and non-competitive with iodide. Thyroid Na+K+ ATPase activity, a major mechanism to drive I influx, decreased by 40% after the cells were exposed to 5 mmol/l H2O2 for 10 min. H2O2 enhanced I efflux only when Ca2+ was present in the medium. The mechanism of an enhanced I efflux by H2O2 appears to be mediated through the elevation of free cytosolic Ca2+ concentration. Our data indicate that H2O2 can affect I transport by inhibiting I influx and enhancing I efflux.

Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide.

PubMed

Juffermans, L J M; Dijkmans, P A; Musters, R J P; Visser, C A; Kamp, O

2006-10-01

In the present study, we addressed the interactions among ultrasound, microbubbles, and living cells as well as consequent arising bioeffects. We specifically investigated whether hydrogen peroxide (H(2)O(2)) is involved in transient permeabilization of cell membranes in vitro after ultrasound exposure at low diagnostic power, in the presence of stable oscillating microbubbles, by measuring the generation of H(2)O(2) and Ca(2+) influx. Ultrasound, in the absence or presence of SonoVue microbubbles, was applied to H9c2 cells at 1.8 MHz with a mechanical index (MI) of 0.1 or 0.5 during 10 s. This was repeated every minute, for a total of five times. The production of H(2)O(2) was measured intracellularly with CM-H(2)DCFDA. Cell membrane permeability was assessed by measuring real-time changes in intracellular Ca(2+) concentration with fluo-4 using live-cell fluorescence microscopy. Ultrasound, in the presence of microbubbles, caused a significant increase in intracellular H(2)O(2) at MI 0.1 of 50% and MI 0.5 of 110% compared with control (P < 0.001). Furthermore, we found increases in intracellular Ca(2+) levels at both MI 0.1 and MI 0.5 in the presence of microbubbles, which was not detected in the absence of extracellular Ca(2+). In addition, in the presence of catalase, Ca(2+) influx immediately following ultrasound exposure was completely blocked at MI 0.1 (P < 0.01) and reduced by 50% at MI 0.5 (P < 0.001). Finally, cell viability was not significantly affected, not even 24 h later. These results implicate a role for H(2)O(2) in transient permeabilization of cell membranes induced by ultrasound-exposed microbubbles.

Evasion of affinity-based selection in germinal centers by Epstein-Barr virus LMP2A.

PubMed

Minamitani, Takeharu; Yasui, Teruhito; Ma, Yijie; Zhou, Hufeng; Okuzaki, Daisuke; Tsai, Chiau-Yuang; Sakakibara, Shuhei; Gewurz, Benjamin E; Kieff, Elliott; Kikutani, Hitoshi

2015-09-15

Epstein-Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell-specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.

Mesoporous silica nanoparticle-based substrates for cell directed delivery of Notch signalling modulators to control myoblast differentiation

NASA Astrophysics Data System (ADS)

Böcking, Dominique; Wiltschka, Oliver; Niinimäki, Jenni; Shokry, Hussein; Brenner, Rolf; Lindén, Mika; Sahlgren, Cecilia

2014-01-01

Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery.Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery. Electronic supplementary information (ESI) available: (1) Particle characterization. (2) Immunohistochemistry and SEM analyses of C2C12 cells grown on films for 3, 6, 24 and 72 h. Light microscopy and WST1 analyses of cells grown on cover slips and films for 6, 24 and 72 h (3) Quantification of protein levels of C2C12 cells differentiating on cover slips versus MSN films. (4) Stability of MSN films in biological solution and the influence on cell viability. (5) Cell internalization of particles from MSN films and intracellular drug release at 12 and 24 h (6) Cell internalization and intracellular DiI release of MSNs from (3Dtro®) fiber scaffolds impregnated with MSNs. See DOI: 10.1039/c3nr04022d

Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits.

PubMed

Kang, Sung Koo; Yi, Kye Sook; Kwon, Nyoun Soo; Park, Kwang-Hyun; Kim, Uh-Hyun; Baek, Kwang Jin; Im, Mie-Jae

2004-08-27

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

Toxicological evaluation of some Malaysian locally processed raw food products.

PubMed

Sharif, R; Ghazali, A R; Rajab, N F; Haron, H; Osman, F

2008-01-01

Malaysian locally processed raw food products are widely used as main ingredients in local cooking. Previous studies showed that these food products have a positive correlation with the incidence of cancer. The cytotoxicity effect was evaluated using MTT assay (3-(4,5-dimetil-2-thiazolil)-2,5-diphenyl-2H-tetrazolium bromide) against Chang liver cells at 2000 microg/ml following 72 h incubation. Findings showed all methanol extracts caused a tremendous drop in the percentage of cell viability at 2000 microg/ml (shrimp paste - 41.69+/-3.36%, salted fish - 37.2+/-1.06%, dried shrimp - 40.32+/-1.8%, p<0.05). To detect DNA damage in a single cell, alkaline Comet Assay was used. None of the extracts caused DNA damage to the Chang liver cells at 62.5 microg/ml following 24 h incubation, as compared to the positive control, hydrogen peroxide (tail moment - 9.50+/-1.50; tail intensity - 30.50+/-2.50). Proximate analysis which was used for the evaluation of macronutrients in food showed that shrimp paste did not comply with the protein requirement (<25%) as in Food Act 1983. Salt was found in every sample with the highest percentage being detected in shrimp paste which exceeded 20%. Following heavy metal analysis (arsenic, cadmium, lead and mercury), arsenic was found in every sample with dried shrimps showing the highest value as compared to the other samples (6.16 mg/kg). In conclusion, several food extracts showed cytotoxic effect but did not cause DNA damage against Chang liver cells. Salt was found as the main additive and arsenic was present in every sample, which could be the probable cause of the toxicity effects observed.

Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells.

PubMed

Son, M-Y; Sim, H; Son, Y S; Jung, K B; Lee, M-O; Oh, J-H; Chung, S-K; Jung, C-R; Kim, J

2017-12-01

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids. © 2017 British Neuropathological Society.

Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

PubMed

Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed; Miller, Barbara A; Melendez, J Andrés

2016-02-19

As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Derivation, characterization and differentiation of a new human embryonic stem cell line from a Chinese hatched blastocyst assisted by a non-contact laser system.

PubMed

Wu, Rongrong; Xu, Chenming; Jin, Fan; Tan, Zhou; Gu, Bin; Chen, Liangbiao; Yao, Xing; Zhang, Ming

2010-08-01

Currently worldwide attention has focused on the derivation of human embryonic stem cells (hESCs) for future therapeutic medicine. However, the majority of existing hESCs are directly or indirectly exposed to non-human materials during their derivation and/or propagation, which greatly restrict their therapeutic potential. Besides the efforts to improve culture systems, the derivation procedure, especially blastocyst manipulation, needs to be optimized. We adopted a non-contact laser-assisted hatching system in combination with sequential culture process to obtain hatched blastocysts as materials for hESC derivation, and derived a hESC line ZJUhES-1 of a Chinese population without exposure to any non-human materials during blastocyst manipulation. ZJUhES-1 satisfies the criteria of pluripotent hESCs: typically morphological characteristics; the expression of alkaline phosphatase, human telomerase reverse transcriptase and multiple hESC-specific markers including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, Nanog, Rex-1, Sox-2, UTF-1, Connexins 43 and 45, TERF-1 and TERF-2, Glut-1, BCRP-1/ABCG-2, GDF3, LIN28, FGF4, Thy-1, Cripto1/TDGF1, AC133 as well as SMAD1/2/3/5; extended proliferative capacity; maintenance of a stable male karyotype after long-term cultivation; and robust multiple-lineage developmental potentials both in vivo and in vitro. Moreover, ZJUhES-1 has distinct identity revealed from DNA fingerprinting. Our xeno-free blastocyst manipulation procedure may promote the progression toward clinical-grade hESC derivation. © 2010 The Authors. Human Cell © 2010 Japan Human Cell Society.

Anolyte recycling enhanced bioelectricity generation of the buffer-free single-chamber air-cathode microbial fuel cell.

PubMed

Ren, Yueping; Chen, Jinli; Shi, Yugang; Li, Xiufen; Yang, Na; Wang, Xinhua

2017-11-01

Anolyte acidification is an inevitable restriction for the bioelectricity generation of buffer-free microbial fuel cells (MFCs). In this work, acidification of the buffer-free KCl anolyte has been thoroughly eliminated through anolyte recycling. The accumulated HCO 3 - concentration in the recycled KCl anolyte was above 50mM, which played as natural buffer and elevated the anolyte pH to above 8. The maximum power density (P max ) increased from 322.9mWm -2 to 527.2mWm -2 , which is comparable with the phosphate buffered MFC. Besides Geobacter genus, the gradually increased anolyte pH and conductivity induced the growing of electrochemically active Geoalkalibacter genus, in the anode biofilm. Anolyte recycling is a feasible strategy to strengthen the self-buffering capacity of buffer-free MFCs, thoroughly eliminate the anolyte acidification and prominently enhance the electric power. Copyright © 2017 Elsevier Ltd. All rights reserved.

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

PubMed

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimizu, Masahito; Department of Medicine, Gifu University School of Medicine, Gifu 501-1194; Deguchi, Atsuko

The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2,more » HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 {mu}g/ml of EGCG (the IC{sub 50} concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 {mu}g/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-{beta}2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.« less

Preparation of Rhodium(III) complexes with 2(1H)-quinolinone derivatives and evaluation of their in vitro and in vivo antitumor activity.

PubMed

Lu, Xing; Wu, Yi-Ming; Yang, Jing-Mei; Ma, Feng-E; Li, Liang-Ping; Chen, Sheng; Zhang, Ye; Ni, Qing-Ling; Pan, Ying-Ming; Hong, Xue; Peng, Yan

2018-05-10

A series of 2(1H)-quinolinone derivatives and their rhodium (III) complexes were designed and synthesized. All the rhodium (III) complexes exhibited higher in vitro cytotoxicity for Hep G2, HeLa 229, MGC80-3, and NCI-H460 human tumor cell lines than their ligands and cisplatin, and among them complex 9 was found to be selectively cytotoxic to tumor cells. Further investigation revealed that complex 9 caused cell cycle arrest at the G2/M phase and induced apoptosis, and inhibited the proliferation of Hep G2 cells by impeding the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream enzymes. Complex 9 also up-regulated the proapoptotic proteins Bak, Bax, and Bim, which altogether activated caspase-3/9 to initiate cell apoptosis. Notably, complex 9 effectively inhibited tumor growth in the NCI-H460 xenograft mouse model with less adverse effect than cisplatin. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A human systemic lupus erythematosus-related anti-cardiolipin/single-stranded DNA autoantibody is encoded by a somatically mutated variant of the developmentally restricted 51P1 V[sub H] gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Es, J.H.; Aanstoot, H.; Gmelig-Meyling, F.H.J.

1992-09-15

The authors report the Ig H and L chain V region sequences from the cDNAs encoding a monoclonal human IgG anti-cardiolipin/ssDNA autoantibody (R149) derived from a patient with active SLE. Comparison with the germ-line V-gene repertoire of this patient revealed that R149 likely arose as a consequence of an Ag-driven selection process. The Ag-binding portions of the V regions were characterized by a high number of arginine residues, a property that has been associated with anti-dsDNA autoantibodies from lupus-prone mice and patients with SLE. The V[sub H] gene encoding autoantibody R149 was a somatically mutated variant of the 51P1 genemore » segment, which is frequently associated with the restricted fetal B cell repertoire, malignant CD5 B cells, and natural antibodies. These data suggest that in SLE patients a common antigenic stimulus may evoke anti-DNA and anti-cardiolipin autoantibodies and provide further evidence that a small set of developmentally restricted V[sub H] genes can give rise to disease-associated autoantibodies through Ag-selected somatic mutations. 42 refs., 5 figs.« less

Characterizing Reactive Flow Paths in Fractured Cement

NASA Astrophysics Data System (ADS)

Wenning, Q. C.; Huerta, N. J.; Hesse, M. A.; Bryant, S. L.

2011-12-01

Geologic carbon sequestration can be a viable method for reducing anthropogenic CO2 flux into the atmosphere. However, the technology must be economically feasible and pose acceptable risk to stakeholders. One key risk is CO2 leakage out of the storage reservoir. Potential driving forces for leakage are the overpressure due to CO2 injection and the buoyancy of free phase CO2. Potential hazards of leakage are contamination of Underground Sources of Drinking Water or the atmosphere and would be deemed an unacceptable risk. Wells potentially provide a fast path for leakage from the reservoir. While the well's cement casing is reactive with CO2 and CO2-saturated brine, the low cement matrix permeability and slow diffusion rate make it unlikely that CO2 will escape through a properly constructed wellbore. However, highly permeable fractures with micrometer scale apertures can occur in cement casings. Reactions that occur in the flow in these fractures can either be self-limiting or self-enhancing. Therefore, understanding the reactive flow is critical to understanding of leakage evolution through these fractures. The goal of our work is to characterize the modification of the flow paths in the fracture due to reaction with acidic brine. With this aim we have characterized both the initial flow path of un-reactive flow and the final flow path after introduction of low-pH acid along the same fracture. Class H cement cores 3-6 cm in length and 2.5 cm diameter are created and a single natural and unique fracture is produced in each core using the Brazilian method. Our experimental fluid is injected at a constant rate into the cement core housed in a Hassler Cell under confining pressure. A solution of red dye and deionized water is pumped through the fracture to stain the un-reactive flow paths. Deionized water is then pumped through the core to limit diffusion of the dye into non-flowing portions of the fracture. After staining the initial flow path, low pH water due to hydrochloric acid (HCL), is pumped through the core at the same rate as the dye. The low pH water is used as a proxy for acidic CO2-saturated brine. Both staining from the un-reactive dye and acid produce visible permanent color alterations on the cement fracture plane. Results show that nearly the entire fracture width is stained by the red dye, with only a few asperities un-dyed. However the low pH HCl forms restricted reacted channels that are a subset of the area open to un-reactive flow, occupying only 10-50% of the entire fracture width. Low pH HCl is believed to be the driving force for the reaction that causes channeling. As acid flows through the fracture, calcium is stripped from the low pH high velocity flow front and precipitates along of the edges of the channel where pH is higher due to the lower flow velocities outside the channel. It is hypothesized that this mineral precipitation restricts the flow into localized channels within the plane of fractures having apertures of tens of micrometers. Reactions restrict the flow path to a smaller fraction of the surface, which may be an indication of self-limiting behavior.

Intermediate frequency magnetic field generated by a wireless power transmission device does not cause genotoxicity in vitro.

PubMed

Shi, Dejing; Zhu, Chunbo; Lu, Rengui; Mao, Shitong; Qi, Yanhua

2014-10-01

The aim of this study was to evaluate effects of intermediate frequency magnetic fields (IFMF) generated by a wireless power transmission (WPT) based on magnetic resonance from the perspective of cellular genotoxicity on cultured human lens epithelial cells (HLECs). We evaluated the effects of exposure to 90 kHz magnetic fields at 93.36 µT on cellular genotoxicity in vitro for 2 and 4 h. The magnetic flux density is approximately 3.5 times higher than the reference level recommended by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. For assessment of genotoxicity, we studied cellular proliferation, apoptosis and DNA damage by Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis, alkaline comet assay and phosphorylated histone H2AX (γH2AX) foci formation test. We did not detect any effect of a 90 kHz IFMF generated by WPT based on magnetic resonance on cell proliferation, apoptosis, comet assay, and γH2AX foci formation test. Our results indicated that exposure to 90 kHz IFMF generated by WPT based on magnetic resonance at 93.36 µT for 2 and 4 h does not cause detectable cellular genotoxicity. © 2014 Wiley Periodicals, Inc.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

PubMed

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Trimer procyanidin oligomers contribute to the protective effects of cinnamon extracts on pancreatic β-cells in vitro

PubMed Central

Sun, Peng; Wang, Ting; Chen, Lu; Yu, Bang-wei; Jia, Qi; Chen, Kai-xian; Fan, Hui-min; Li, Yi-ming; Wang, He-yao

2016-01-01

Aim: Cinnamon extracts rich in procyanidin oligomers have shown to improve pancreatic β-cell function in diabetic db/db mice. The aim of this study was to identify the active compounds in extracts from two species of cinnamon responsible for the pancreatic β-cell protection in vitro. Methods: Cinnamon extracts were prepared from Cinnamomum tamala (CT-E) and Cinnamomum cassia (CC-E). Six compounds procyanidin B2 (cpd1), (−)-epicatechin (cpd2), cinnamtannin B1 (cpd3), procyanidin C1 (cpd4), parameritannin A1 (cpd5) and cinnamtannin D1 (cpd6) were isolated from the extracts. INS-1 pancreatic β-cells were exposed to palmitic acid (PA) or H2O2 to induce lipotoxicity and oxidative stress. Cell viability and apoptosis as well as ROS levels were assessed. Glucose-stimulated insulin secretion was examined in PA-treated β-cells and murine islets. Results: CT-E, CC-E as well as the compounds, except cpd5, did not cause cytotoxicity in the β-cells up to the maximum dosage using in this experiment. CT-E and CC-E (12.5–50 μg/mL) dose-dependently increased cell viability in both PA- and H2O2-treated β-cells, and decreased ROS accumulation in H2O2-treated β-cells. CT-E caused more prominent β-cell protection than CC-E. Furthermore, CT-E (25 and 50 μg/mL) dose-dependently increased glucose-stimulated insulin secretion in PA-treated β-cells and murine islets, but CC-E had little effect. Among the 6 compounds, trimer procyanidins cpd3, cpd4 and cpd6 (12.5–50 μmol/L) dose-dependently increased the cell viability and decreased ROS accumulation in H2O2-treated β-cells. The trimer procyanidins also increased glucose-stimulated insulin secretion in PA-treated β-cells. Conclusion: Trimer procyanidins in the cinnamon extracts contribute to the pancreatic β-cell protection, thus to the anti-diabetic activity. PMID:27238208

Glutaredoxin 2 prevents H(2)O(2)-induced cell apoptosis by protecting complex I activity in the mitochondria.

PubMed

Wu, Hongli; Xing, Kuiyi; Lou, Marjorie F

2010-10-01

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200muM hydrogen peroxide (H(2)O(2)) for 24h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H(2)O(2)-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H(2)O(2) treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H(2)O(2)-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H(2)O(2)-induced apoptosis is likely associated with its ability to preserve complex I. Published by Elsevier B.V.

Expression of mutant Sftpcin murine alveolar epithelia drives spontaneous lung fibrosis.

PubMed

Nureki, Shin-Ichi; Tomer, Yaniv; Venosa, Alessandro; Katzen, Jeremy; Russo, Scott J; Jamil, Sarita; Barrett, Matthew; Nguyen, Vivian; Kopp, Meghan; Mulugeta, Surafel; Beers, Michael F

2018-06-19

Epithelial cell dysfunction is postulated as an important component in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Mutations in the Surfactant Protein C [SP-C] gene [SFTPC], an alveolar type 2 (AT2) cell restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knock-in mouse model capable of regulated expression of an IPF-associated Isoleucine to Threonine substitution at codon 73 [I73T] in Sftpc (SP-CI73T). Tamoxifen treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA contents of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGFB1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, a-SMA positive cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis strengthening the role of AT2 dysfunction as a key upstream driver of IPF pathogenesis.

Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors

PubMed Central

Doulatov, Sergei; Vo, Linda T.; Chou, Stephanie S.; Kim, Peter G.; Arora, Natasha; Li, Hu; Hadland, Brandon K.; Bernstein, Irwin D.; Collins, James J.; Zon, Leonard I.; Daley, George Q.

2013-01-01

Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells. PMID:24094326

Design, calibration and application of broad-range optical nanosensors for determining intracellular pH.

PubMed

Søndergaard, Rikke V; Henriksen, Jonas R; Andresen, Thomas L

2014-12-01

Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.

3H-1,2-dithiole-3-thione protects retinal pigment epithelium cells against Ultra-violet radiation via activation of Akt-mTORC1-dependent Nrf2-HO-1 signaling.

PubMed

Li, Ke-Ran; Yang, Su-Qing; Gong, Yi-Qing; Yang, Hong; Li, Xiu-Miao; Zhao, Yu-Xia; Yao, Jin; Jiang, Qin; Cao, Cong

2016-05-06

Excessive UV radiation and reactive oxygen species (ROS) cause retinal pigment epithelium (RPE) cell injuries. Nrf2 regulates transcriptional activation of many anti-oxidant genes. Here, we tested the potential role of 3H-1,2-dithiole-3-thione (D3T) against UV or ROS damages in cultured RPE cells (both primary cells and ARPE-19 line). We showed that D3T significantly inhibited UV-/H2O2-induced RPE cell death and apoptosis. UV-stimulated ROS production was dramatically inhibited by D3T pretreatment. D3T induced Nrf2 phosphorylation in cultured RPE cells, causing Nrf2 disassociation with KEAP1 and its subsequent nuclear accumulation. This led to expression of antioxidant response elements (ARE)-dependent gene heme oxygenase-1 (HO-1). Nrf2-HO-1 activation was required for D3T-mediated cytoprotective effect. Nrf2 shRNA knockdown or S40T dominant negative mutation as well as the HO-1 inhibitor Zinc protoporphyrin (ZnPP) largely inhibited D3T's RPE cytoprotective effects against UV radiation. Yet, exogenous overexpression Nrf2 enhanced D3T's activity in RPE cells. Further studies showed that D3T activated Akt/mTORC1 in cultured RPE cells. Akt-mTORC1 inhibitors, or Akt1 knockdown by shRNA, not only inhibited D3T-induced Nrf2-HO-1 activation, but also abolished the RPE cytoprotective effects. In vivo, D3T intravitreal injection protected from light-induced retinal dysfunctions in mice. Thus, D3T protects RPE cells from UV-induced damages via activation of Akt-mTORC1-Nrf2-HO-1 signaling axis.

Long Non-Coding RNA H19 Protects H9c2 Cells against Hypoxia-Induced Injury by Targeting MicroRNA-139.

PubMed

Gong, Li-Cheng; Xu, Hai-Ming; Guo, Gong-Liang; Zhang, Tao; Shi, Jing-Wei; Chang, Chang

2017-01-01

Acute myocardial infarction (AMI) occurs when blood supply to the heart is diminished (ischemia) for long time; ischemia is primarily caused due to hypoxia. The present study evaluated the effects of long non-coding RNA H19 on hypoxic rat H9c2 cells and mouse HL-1 cells. Hypoxic injury was confirmed by measuring cell viability, migration and invasion, and apoptosis using MTT, Transwell and flow cytometry assays, respectively. H19 expression after hypoxia was estimated by qRT-PCR. We then measured the effects of non-physiologically expressed H19, knockdown of miR-139 with or without H19 silence, and abnormally expressed Sox8 on hypoxia-induced H9c2 cells. Moreover, the interacted miRNA for H19 and downstream target gene were virtually screened and verified. The involved signaling pathways and the effects of abnormally expressed H19 on contractility of HL-1 cells were explored via Western blot analysis. Hypoxia induced decreases of cell viability, migration and invasion, increase of cell apoptosis and up-regulation of H19. Knockdown of H19 increased hypoxia-induced injury in H9c2 cells. H19 acted as a sponge for miR-139 and H19 knockdown aggravated hypoxia-induced injury by up-regulating miR-139. Sox8 was identified as a target of miR-139, and its expression was negatively regulated by miR-139. The mechanistic studies revealed that overexpression of Sox8 might decrease hypoxia-induced cell injury by activating the PI3K/AKT/mTOR pathway and MAPK. Besides, H19 promoted contractility of HL-1 cells. These findings suggest that H19 alleviates hypoxia-induced myocardial cell injury by miR-139-mediated up-regulation of Sox8, along with activation of the PI3K/AKT/mTOR pathway and MAPK. © 2017 The Author(s). Published by S. Karger AG, Basel.

The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle.

PubMed Central

Johnston, L H; Eberly, S L; Chapman, J W; Araki, H; Sugino, A

1990-01-01

Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously. In this paper, one of them, dbf2, is characterized in detail. The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined. This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids). The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases. DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene. Its first function was completed very near to initiation of DNA synthesis. However, DNA synthesis in the mutant was only delayed at 37 degrees C, and the cells blocked in nuclear division. Consistent with this finding, the execution point occurred about 1 h after DNA synthesis, and the nuclear morphology of the mutant at the restrictive temperature was that of cells blocked in late nuclear division. DBF2 is therefore likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division. Images PMID:2181271

Differential effects of antifungal agents on expression of genes related to formation of Candida albicans biofilms.

PubMed

Chatzimoschou, Athanasios; Simitsopoulou, Maria; Antachopoulos, Charalampos; Walsh, Thomas J; Roilides, Emmanuel

2016-01-01

The purpose of this study was to analyse specific molecular mechanisms involved in the intrinsic resistance of C. albicans biofilms to antifungals. We investigated the transcriptional profile of three genes (BGL2, SUN41, ECE1) involved in Candida cell wall formation in response to voriconazole or anidulafungin after the production of intermediate and mature biofilms. C. albicans M61, a well-documented biofilm producer strain, was used for the development of intermediate (12 h and 18 h) and completely mature biofilms (48 h). After exposure of cells from each biofilm growth mode to voriconazole (128 and 512 mg l(-1)) or anidulafungin (0.25 and 1 mg l(-1)) for 12-24 h, total RNA samples extracted from biofilm cells were analysed by RT-PCR. The voriconazole and anidulafungin biofilm MIC was 512 and 0.5 mg l(-1) respectively. Anidulafungin caused significant up-regulation of SUN41 (3.7-9.3-fold) and BGL2 (2.2-2.8 fold) in intermediately mature biofilms; whereas, voriconazole increased gene expression in completely mature biofilms (SUN41 2.3-fold, BGL2 2.1-fold). Gene expression was primarily down-regulated by voriconazole in intermediately, but not completely mature biofilms. Both antifungals caused down-regulation of ECE1 in intermediately mature biofilms. © 2015 Blackwell Verlag GmbH.

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells

PubMed Central

Lin, C.; Agnes, J. T.; Behrens, N.; Tagawa, Y.; Gershwin, L. J.; Corbeil, L. B.

2016-01-01

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo. PMID:26859677

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

PubMed

Lin, C; Agnes, J T; Behrens, N; Shao, M; Tagawa, Y; Gershwin, L J; Corbeil, L B

2016-01-01

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2) and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

Benzo[a]pyrene-7,8-dihydrodiol promotes checkpoint activation and G2/M arrest in human bronchoalveolar carcinoma H358 cells.

PubMed

Caino, M Cecilia; Oliva, Jose L; Jiang, Hao; Penning, Trevor M; Kazanietz, Marcelo G

2007-03-01

Polycyclic aromatic hydrocarbons (PAHs) are potent carcinogens that require metabolic activation inside cells. The proximate carcinogens PAH-diols can be converted to o-quinones by aldo-keto reductases (AKRs) or to diol-epoxides by cytochrome P450 (P450) enzymes. We assessed the effect of benzo[a]pyrene-7,8-dihydrodiol (BPD) on proliferation in p53-null bronchoalveolar carcinoma H358 cells. BPD treatment led to a significant inhibition of proliferation and arrest in G2/M in H358 cells. The relative contribution of the AKR and P450 pathways to cell cycle arrest was assessed. Overexpression of AKR1A1 did not affect cell proliferation or cell cycle progression, and benzo[a]pyrene-7,8-dione did not cause any noticeable effect on cell growth, suggesting that AKR1A1 metabolic products were not involved in the antiproliferative effect of BPD. On the other hand, blockade of P450 induction or inhibition of P450 activity greatly impaired the effect of BPD. Moreover, P450 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly enhanced the antiproliferative effect of BPD. Mechanistic studies revealed that BPD caused a DNA damage response, Chk1 activation, and accumulation of phospho-Cdc2 (Tyr15) in H358 cells, effects that were impaired by an ataxia-telangectasia mutated (ATM)/ATM-related (ATR) inhibitor. Similar results were observed in human bronchoepithelial BEAS-2B cells, arguing for analogous mechanisms in tumorigenic and immortalized nontumorigenic cells lacking functional p53. Our data suggest that a p53-independent pathway operates in lung epithelial cells in response to BPD that involves P450 induction and subsequent activation of the ATR/ATM/Chk1 damage check-point pathway and cell cycle arrest in G2/M.

HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana

Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24 h and then treated with HBCDD + hCG for additional 2 h. Results showed that HBCDD caused a sustained reduction in ATPmore » level after 24 h of exposure, which persisted after additional 2-hour treatment with HBCDD + hCG. cAMP and androgen accumulations measured after 2 h of HBCDD + hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30 kDa steroidogenic acute regulatory protein (StAR) after HBCDD + hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells. - Highlights: • HBCDD causes a sustained reduction in ΔΨm and ATP level in Leydig cells. • Prolonged HBCDD exposure decreases hCG-supported steroidogenesis in Leydig cells. • HBCDD targets StAR, HSD17β and CYP11A1 in Leydig cells.« less

Upregulation of NAD(P)H:Quinone Oxidoreductase By Radiation Potentiates the Effect of Bioreductive β-Lapachone on Cancer Cells1

PubMed Central

Choi, Eun K; Terai, Kaoru; Ji, In-Mi; Kook, Yeon H; Park, Kyung H; Oh, Eun T; Griffin, Robert J; Lim, Byung U; Kim, Jin-Seok; Lee, Doo S; Boothman, David A; Loren, Melissa; Song, Chang W; Park, Heon Joo

2007-01-01

We found that β-lapachone (β-lap), a novel bioreductive drug, caused rapid apoptosis and clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by β-lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(P)H:quinone oxido-reductase (NQO1), and also by siRNA for NQO1, demonstrating that NQO1-induced bioreduction of β-lap is an essential step in β-lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of NQO1, thereby increasing NQO1-mediated β-lap-induced cell deaths. Although the direct cause of β-lap-induced apoptosis is not yet clear, β-lap treatment reduced the expression of p53 and NF-κB, whereas it increased cytochrome C release, caspase-3 activity, and γH2AX foci formation. Importantly, β-lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that β-lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by β-lap treatment. This is the first study to demonstrate that combined radiotherapy and β-lap treatment can have a significant effect on human tumor xenografts. PMID:17786182

β-sitosterol induces G1 arrest and causes depolarization of mitochondrial membrane potential in breast carcinoma MDA-MB-231 cells

PubMed Central

2013-01-01

Backgrounds It is suggested that dietary phytosterols, such as β-sitosterol (ST), have cancer chemopreventive effects; however, studies are limited to support such claims. Here, we evaluated the efficacy of ST on three different human cancer cell lines including skin epidermoid carcinoma A431 cells, lung epithelial carcinoma A549 cells and breast adenocarcinoma MDA-MB-231. Methods Cell growth assay, cell cycle analysis, FACS, JC-1 staining, annexin V staining and immunoblotting were used to study the efficacy of ST on cancer cells. Results ST (30–90 μM) treatments for 48 h and 72 h did not show any significant effect on cell growth and death in A431 cells. Whereas similar ST treatments moderately inhibited the growth of A549 cells by up to 13% (p ≤ 0.05) in 48 h and 14% (p ≤ 0.05-0.0001) in 72 h. In MDA-MB-231 cells, ST caused a significant dose-dependent cell growth inhibition by 31- 63% (p ≤ 0.0001) in 48 h and 40-50% (p ≤ 0.0001) in 72 h. While exploring the molecular changes associated with strong ST efficacy in breast cancer cells, we observed that ST induced cell cycle arrest as well as cell death. ST caused G0/G1 cell cycle arrest which was accompanied by a decrease in CDK4 and cyclin D1, and an increase in p21/Cip1and p27/Kip1 protein levels. Further, cell death effect of ST was associated with induction of apoptosis. ST also caused the depolarization of mitochondrial membrane potential and increased Bax/Bcl-2 protein ratio. Conclusions These results suggest prominent in vitro anti-proliferative and pro-apoptotic effects of ST in MDA-MB-231 cells. This study provides valuable insight into the chemopreventive efficacy and associated molecular alterations of ST in breast cancer cells whereas it had only moderate efficacy on lung cancer cells and did not show any considerable effect on skin cancer cells. These findings would form the basis for further studies to understand the mechanisms and assess the potential utility of ST as a cancer chemopreventive agent against breast cancer. PMID:24160369

Repeating patterns of sleep restriction and recovery: Do we get used to it?

PubMed

Simpson, Norah S; Diolombi, Moussa; Scott-Sutherland, Jennifer; Yang, Huan; Bhatt, Vrushank; Gautam, Shiva; Mullington, Janet; Haack, Monika

2016-11-01

Despite its prevalence in modern society, little is known about the long-term impact of restricting sleep during the week and 'catching up' on weekends. This common sleep pattern was experimentally modeled with three weeks of 5 nights of sleep restricted to 4h followed by two nights of 8-h recovery sleep. In an intra-individual design, 14 healthy adults completed both the sleep restriction and an 8-h control condition, and the subjective impact and the effects on physiological markers of stress (cortisol, the inflammatory marker IL-6, glucocorticoid receptor sensitivity) were assessed. Sleep restriction was not perceived to be subjectively stressful and some degree of resilience or resistance to the effects of sleep restriction was observed in subjective domains. In contrast, physiological stress response systems remain activated with repeated exposures to sleep restriction and limited recovery opportunity. Morning IL-6 expression in monocytes was significantly increased during week 2 and 3 of sleep restriction, and remained increased after recovery sleep in week 2 (p<0.05) and week 3 (p<0.09). Serum cortisol showed a significantly dysregulated 24h-rhythm during weeks 1, 2, and 3 of sleep restriction, with elevated morning cortisol, and decreased cortisol in the second half of the night. Glucocorticoid sensitivity of monocytes was increased, rather than decreased, during the sleep restriction and sleep recovery portion of each week. These results suggest a disrupted interplay between the hypothalamic-pituitary-adrenal and inflammatory systems in the context of repeated exposure to sleep restriction and recovery. The observed dissociation between subjective and physiological responses may help explain why many individuals continue with the behavior pattern of restricting and recovering sleep over long time periods, despite a cumulative deleterious physiological effect. Copyright © 2016 Elsevier Inc. All rights reserved.

Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology

PubMed Central

Saxena, Kapil; Blutt, Sarah E.; Ettayebi, Khalil; Zeng, Xi-Lei; Broughman, James R.; Crawford, Sue E.; Karandikar, Umesh C.; Sastri, Narayan P.; Conner, Margaret E.; Opekun, Antone R.; Graham, David Y.; Qureshi, Waqar; Sherman, Vadim; Foulke-Abel, Jennifer; In, Julie; Kovbasnjuk, Olga; Zachos, Nicholas C.; Donowitz, Mark

2015-01-01

ABSTRACT Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures. PMID:26446608

Mechanistic Target of Rapamycin Is a Novel Molecular Mechanism Linking Folate Availability and Cell Function.

PubMed

Silva, Elena; Rosario, Fredrick J; Powell, Theresa L; Jansson, Thomas

2017-07-01

Folate deficiency has been linked to a wide range of disorders, including cancer, neural tube defects, and fetal growth restriction. Folate regulates cellular function mediated by its involvement in the synthesis of nucleotides, which are needed for DNA synthesis, and its function as a methyl donor, which is critical for DNA methylation. Here we review current data showing that folate sensing by mechanistic target of rapamycin (mTOR) constitutes a novel and distinct pathway by which folate modulates cell functions such as nutrient transport, protein synthesis, and mitochondrial respiration. The mTOR signaling pathway responds to growth factors and changes in nutrient availability to control cell growth, proliferation, and metabolism. mTOR exists in 2 complexes, mTOR complex (mTORC) 1 and mTORC2, which have distinct upstream regulators and downstream targets. Folate deficiency in pregnant mice caused a marked inhibition of mTORC1 and mTORC2 signaling in multiple maternal and fetal tissues, downregulation of placental amino acid transporters, and fetal growth restriction. In addition, folate deficiency in primary human trophoblast (PHT) cells resulted in inhibition of mTORC1 and mTORC2 signaling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells is independent of the accumulation of homocysteine and requires the proton-coupled folate transporter (PCFT; solute carrier 46A1). Furthermore, mTORC1 and mTORC2 regulate trophoblast folate uptake by modulating the cell surface expression of folate receptor α and the reduced folate carrier. These findings, which provide a novel link between folate availability and cell function, growth, and proliferation, may have broad biological significance given the critical role of folate in normal cell function and the multiple diseases that have been associated with decreased or excessive folate availability. Low maternal folate concentrations are linked to restricted fetal growth, and we propose that the underlying mechanisms involve trophoblast mTOR folate sensing resulting in inhibition of mTORC1 and mTORC2 and downregulation of placental amino acid transporters. © 2017 American Society for Nutrition.

pH-Sensitive fusogenic polymer-modified liposomes as a carrier of antigenic proteins for activation of cellular immunity.

PubMed

Yuba, Eiji; Kojima, Chie; Harada, Atsushi; Tana; Watarai, Shinobu; Kono, Kenji

2010-02-01

By modification of liposomes with poly(glycidol) derivatives such as succinylated poly(glycidol) and 3-methylglutarylated poly(glycidol), we have developed functional liposomes that generate fusion ability at mildly acidic pH. We investigated the feasibility of these polymer-modified liposomes as a carrier of antigenic proteins for induction of cellular immunity. These pH-sensitive fusogenic liposomes encapsulating ovalbumin (OVA) were applied to DC2.4 cells, a murine dendritic cell line. Observation with confocal laser scanning microscopy showed that these polymer-modified liposomes were taken up efficiently by the cells, thereafter delivering their contents into the cytosol, probably through fusion with endosomal membranes. Murine bone marrow-derived dendritic cells treated with polymer-modified liposomes encapsulating OVA stimulated CD8-OVA1.3 cells more strongly than OT4H.1D5 cells, indicating that the liposomes induced MHC class I-restricted presentation. Furthermore, administration of the polymer-modified, OVA-loaded liposomes from nasal cavities of mice induced stronger cellular immune responses than the OVA-loaded plain liposomes. Because the ability of the polymer-modified liposomes to activate cellular immunity was comparable to that of Freund's complete adjuvant, which is a widely used adjuvant, they potentially have use in production of efficient vaccines for immunotherapy.

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Notcovich, Cintia; Laboratorio de Farmacologia de Receptores, Catedra de Quimica Medicinal, Departamento de Farmacologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires; Diez, Federico

2010-02-01

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G{sub 11}-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changesmore » in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP {beta}2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or {beta}2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [{sup 3}H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.« less

Recovery from severe H7N9 disease is associated with diverse response mechanisms dominated by CD8+ T cells

PubMed Central

Wang, Zhongfang; Wan, Yanmin; Qiu, Chenli; Quiñones-Parra, Sergio; Zhu, Zhaoqin; Loh, Liyen; Tian, Di; Ren, Yanqin; Hu, Yunwen; Zhang, Xiaoyan; Thomas, Paul G.; Inouye, Michael; Doherty, Peter C.; Kedzierska, Katherine; Xu, Jianqing

2015-01-01

The avian origin A/H7N9 influenza virus causes high admission rates (>99%) and mortality (>30%), with ultimately favourable outcomes ranging from rapid recovery to prolonged hospitalization. Using a multicolour assay for monitoring adaptive and innate immunity, here we dissect the kinetic emergence of different effector mechanisms across the spectrum of H7N9 disease and recovery. We find that a diversity of response mechanisms contribute to resolution and survival. Patients discharged within 2–3 weeks have early prominent H7N9-specific CD8+ T-cell responses, while individuals with prolonged hospital stays have late recruitment of CD8+/CD4+ T cells and antibodies simultaneously (recovery by week 4), augmented even later by prominent NK cell responses (recovery >30 days). In contrast, those who succumbed have minimal influenza-specific immunity and little evidence of T-cell activation. Our study illustrates the importance of robust CD8+ T-cell memory for protection against severe influenza disease caused by newly emerging influenza A viruses. PMID:25967273

Red paprika (Capsicum annuum L.) and its main carotenoids, capsanthin and β-carotene, prevent hydrogen peroxide-induced inhibition of gap-junction intercellular communication.

PubMed

Kim, Ji-Sun; Lee, Woo-Moon; Rhee, Han Cheol; Kim, Suna

2016-07-25

This study was conducted to investigate the protective effect of red paprika extract (RPE) and its main carotenoids, namely, capsanthin (CST) and β-carotene (BCT), on the H2O2-induced inhibition of gap-junction intercellular communication (GJIC) in WB-F344 rat liver epithelial cells (WB cells). We found that pre-treatment with RPE, CST and BCT protected WB cells from H2O2-induced inhibition of GJIC. RPE, CST and BCT not only recovered connexin 43 (Cx43) mRNA expression but also prevented phosphorylation of Cx43 protein by H2O2 treatment. RPE attenuated the phosphorylation of ERK, p38 and JNK, whereas pre-treatment with CST and BCT only attenuated the phosphorylation of ERK and p38 and did not affect JNK in H2O2-treated WB cells. RPE, CST and BCT significantly suppressed the formation of reactive oxygen species (ROS) in H2O2-treated cells compared to untreated WB cells. These results suggest that dietary intake of red paprika might be helpful for lowering the risk of diseases caused by oxidative stress. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Vitamin K3 triggers human leukemia cell death through hydrogen peroxide generation and histone hyperacetylation.

PubMed

Lin, Changjun; Kang, Jiuhong; Zheng, Rongliang

2005-10-01

Vitamin K3 (VK3) is a well-known anticancer agent, but its mechanism remains elusive. In the present study, VK3 was found to simultaneously induce cell death, reactive oxygen species (ROS) generation, including superoxide anion (O2*-) and hydrogen peroxide (H2O2) generation, and histone hyperacetylation in human leukemia HL-60 cells in a concentration- and time-dependent manner. Catalase (CAT), an antioxidant enzyme that specifically scavenges H2O2, could significantly diminish both histone acetylation increase and cell death caused by VK3, whereas superoxide dismutase (SOD), an enzyme that specifically eliminates O2*-, showed no effect on both of these, leading to the conclusion that H2O2 generation, but not O2*- generation, contributes to VK3-induced histone hyperacetylation and cell death. This conclusion was confirmed by the finding that enhancement of VK3-induced H2O2 generation by vitamin C (VC) could significantly promote both the histone hyperacetylation and cell death. Further studies suggested that histone hyperacetylation played an important role in VK3-induced cell death, since sodium butyrate, a histone deacetylase (HDAC) inhibitor, showed no effect on ROS generation, but obviously potentiated VK3-induced histone hyperacetylation and cell death. Collectively, these results demonstrate a novel mechanism for the anticancer activity of VK3, i.e., VK3 induced tumor cell death through H2O2 generation, which then further induced histone hyperacetylation.

Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamashita, Sachiko; Tanaka, Masakazu; Sato, Teruaki

Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h andmore » 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24–72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX, PAR could be a sensitive marker for DNA single- and double-strand breaks. These two molecular markers provide evidence of physiological changes occurring within cells. - Highlights: • Physiological level of poly(ADP-ribose) was determined during mild temperature shift. • Poly(ADP-ribose) level in HeLa and CHO-K1 cells significantly increased. • γH2AX signals increased during culture at 40.5 °C as compared to 37.0 °C. • Poly(ADP-ribose) polymerase inhibitor potentiated γH2AX signals at 40.5 °C. • γH2AX and poly(ADP-ribose) level provide evidence of physiological changes in cells.« less

[Immunogenicity of chimeric gene vaccine Mtb8.4/hIL12].

PubMed

Li, Hui; Li, Rong; Zhong, Sen; Luo, Yue-bei; Ren, Hong; Deng, Cun-liang

2006-09-01

To construct chimeric gene vaccine Mtb8.4/hIL-12, express it in COS-7 cells and study its immunogenicity. Chimeric gene Mtb8.4/hIL-12 was amplified by PCR and cloned into the eukaryotic vector pCI-neo to construct the recombinant plasmid pCI-neo-Mtb8.4/hIL12. After the recombinant plasmid was identified by restriction enzyme digestion analysis, PCR and DNA sequencing, COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12 through cationic liposome. 48 hours later, the expression of mRNA was detected by RT-PCR and the level of hIL-12 in culture supernatant and cell lysates were detected by Western blot. C57BL/6N mice were vaccinated with chimeric gene vaccine Mtb8.4/hIL-12 three times at the interval of 3 weeks each time. Four weeks after the final inoculation, three mice were sacrificed to assess the cytotoxicity of CTLs and response to cytokine. The recombinant plasmid pCI-neo-Mtb8.4/hIL12 was constructed successfully. After COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12, chimeric gene Mtb8.4/hIL12 was expressed in COS-7 cells. The chimeric gene vaccine could induce strong antigen-specific immune response. With the increase of IFN-gamma and IL-2 secretion and the decrease of IL-4 secretion, the cytotoxicity of specific CTLs was heightened. Recombinant plasmid pCI-neo-Mtb8.4/hIL12 has been successfully constructed and expressed in COS-7 cells. The constructed chimeric gene vaccine Mtb8.4/hIL12 is of strong immunogenicity and can obviously induce the cytotoxicity of CTLs.

H2S protects against methionine-induced oxidative stress in brain endothelial cells.

PubMed

Tyagi, Neetu; Moshal, Karni S; Sen, Utpal; Vacek, Thomas P; Kumar, Munish; Hughes, William M; Kundu, Soumi; Tyagi, Suresh C

2009-01-01

Homocysteine (Hcy) causes cerebrovascular dysfunction by inducing oxidative stress. However, to date, there are no strategies to prevent Hcy-induced oxidative damage. Hcy is an H2S precursor formed from methionine (Met) metabolism. We aimed to investigate whether H2S ameliorated Met-induced oxidative stress in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to Met treatment in the presence or absence of NaHS (donor of H2S). Met-induced cell toxicity increased the levels of free radicals in a concentration-dependent manner. Met increased NADPH-oxidase-4 (NOX-4) expression and mitigated thioredxion-1(Trx-1) expression. Pretreatment of bEnd3 with NaHS (0.05 mM) attenuated the production of free radicals in the presence of Met and protected the cells from oxidative damage. Furthermore, NaHS enhanced inhibitory effects of apocynin, N-acetyl-l-cysteine (NAC), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), Nomega-nitro-l-arginine methyl ester (L-NAME) on ROS production and redox enzymes levels induced by Met. In conclusion, the administration of H2S protected the cells from oxidative stress induced by hyperhomocysteinemia (HHcy), which suggested that NaHS/H2S may have therapeutic potential against Met-induced oxidative stress.

H-2 compatibility requirement for virus-specific T-cell-mediated cytolysis. Evaluation of the role of H-2I region and non-H-2 genes in regulating immune response

PubMed Central

1976-01-01

Lymphocytic choriomeningitis virus (LCMV) and ectromelia virus-specific T-cell-mediated cytotoxicity was assayed in various strain combinations using as targets peritoneal macrophages which have been shown to express Ia antigens. Virus-specific cytotoxicity was found only in H-2K- or D-region compatible combinations. I-region compatibility was not necessary nor alone sufficient for lysis. Six different I-region specificities had no obvious effect on the capacity to generate in vivo specific cytotoxicity (expressed in vitro) associated with Dd. Low LCMV- specific cytotoxic activity generated in DBA/2 mice was caused by the non-H-2 genetic background. This trait was inversely related to the infectious virus dose and recessive. Non-H-2 genes, possibly involved in controlling initial spread and multiplication of virus, seem to be, at least in the examples tested, more important in determining virus- specific cytotoxic T-cell activity in spleens than are Ir genes coded in H-2. PMID:1085331

Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

PubMed

Neuhaus, Jochen; Oberbach, Andreas; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2006-05-01

To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

Effects of quercetin on CDK4 mRNA and protein expression in A549 cells infected by H1N1

PubMed Central

WAN, QIAOFENG; WANG, HAO; LIN, YUAN; GU, LIGANG; HAN, MEI; YANG, ZHIWEI; ZHANG, YANLI; MA, RUI; WANG, LI; WANG, ZHISHENG

2013-01-01

This study was conducted to investigate the effects of quercetin on the expression of cyclin-dependent kinase (CDK4) mRNA and protein in A549 lung epithelial tumor cells infected by H1N1. First, the Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to determine H1N1 virulence, quercetin cytotoxicity and inhibition of the cytopathic effect of H1N1 on A549 cells by quercetin. Subsequently, 100 TCID50 H1N1 was used to infect A549 cells for 2 h prior to culture in maintenance media containing 10 mg/l quercetin. After 4, 12, 24 and 48 h of culture, the cells were collected and total RNA and protein were extracted. Fluorescent quantitative polymerase chain reaction and western blot analysis were then performed to assess the expression of CDK4 mRNA and protein. The experiment demonstrated that the TCID50 of H1N1 in A549 cells was 10−4.75, the maximum non-toxic concentration of quercetin in A549 cells was 30–60 mg/l and the minimum effective concentration of quercetin for the inhibition of the H1N1 cytopathic effect on A549 cells was 10 mg/l. The results indicated that quercetin may significantly inhibit CDK4 mRNA and protein overexpression caused by H1N1 within 4–48 h. In conclusion, quercetin may protect against H1N1 infection by effectively reducing the mRNA and protein expression of CDK4 caused by H1N1 infection. PMID:24649026

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival.

PubMed

Gingery, Anne; Bradley, Elizabeth; Shaw, Aubie; Oursler, Merry Jo

2003-05-01

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival. Copyright 2003 Wiley-Liss, Inc.

A flexible all-inorganic fuel cell membrane with conductivity above Nafion, and durable operation at 150 °C

NASA Astrophysics Data System (ADS)

Ansari, Y.; Tucker, T. G.; Huang, W.; Klein, I. S.; Lee, S.-Y.; Yarger, J. L.; Angell, C. A.

2016-01-01

The search for fuel cell membranes has focused on carbon backbone polymers, among which Nafion seems to best survive the most severe of the degradation mechanisms - attack by peroxide radicals. Less attention has been given to inorganic membranes because of their generally inflexible nature and lower conductivity, though some SiO2-Nafion composites have shown improved properties. Nafion dominates, despite needing hydration, which then restricts operation to below 100 °C (so CO poisoning problems persist). Described herein is a low cost, flexible, and all-inorganic fiberglass reinforced gel membrane with conductivity exceeding that of Nafion at any temperature above 60 °C. Using Teflon fuel cells, maximum currents > 1 Acm-2 and OCV of 1.03 V at 150 °C are demonstrated. No detectable loss of cell potential was observed over 24 h during 50 mAcm-2 constant current operation at 120 °C while, at 150 °C and maximum power, the degradation rate is intermediate among other high conductivity H3PO4-PBI type membranes. The structure of the membrane is deduced, mainly from 29Si solid state-NMR. The -115 ppm resonance, which is extreme for Q4 Si(O) structures, identifies a zeolite-like SiO2 network, which is ;floppy;. 31P and 1H NMR establish nano-permeating H3PO4 as the source of the exceptional conductivity.

Disaggregation of Amylin Aggregate by Novel Conformationally Restricted Aminobenzoic Acid containing α/β and α/γ Hybrid Peptidomimetics

PubMed Central

Paul, Ashim; Kalita, Sourav; Kalita, Sujan; Sukumar, Piruthivi; Mandal, Bhubaneswar

2017-01-01

Diabetes has emerged as a threat to the current world. More than ninety five per cent of all the diabetic population has type 2 diabetes mellitus (T2DM). Aggregates of Amylin hormone, which is co-secreted with insulin from the pancreatic β-cells, inhibit the activities of insulin and glucagon and cause T2DM. Importance of the conformationally restricted peptides for drug design against T2DM has been invigorated by recent FDA approval of Symlin, which is a large conformationally restricted peptide. However, Symlin still has some issues including solubility, oral bioavailability and cost of preparation. Herein, we introduced a novel strategy for conformationally restricted peptide design adopting a minimalistic approach for cost reduction. We have demonstrated efficient inhibition of amyloid formation of Amylin and its disruption by a novel class of conformationally restricted β-sheet breaker hybrid peptidomimetics (BSBHps). We have inserted β, γ and δ -aminobenzoic acid separately into an amyloidogenic peptide sequence, synthesized α/β, α/γ and α/δ hybrid peptidomimetics, respectively. Interestingly, we observed the aggregation inhibitory efficacy of α/β and α/γ BSBHps, but not of α/δ analogues. They also disrupt existing amyloids into non-toxic forms. Results may be useful for newer drug design against T2DM as well as other amyloidoses and understanding amyloidogenesis. PMID:28054630

Disaggregation of Amylin Aggregate by Novel Conformationally Restricted Aminobenzoic Acid containing α/β and α/γ Hybrid Peptidomimetics

NASA Astrophysics Data System (ADS)

Paul, Ashim; Kalita, Sourav; Kalita, Sujan; Sukumar, Piruthivi; Mandal, Bhubaneswar

2017-01-01

Diabetes has emerged as a threat to the current world. More than ninety five per cent of all the diabetic population has type 2 diabetes mellitus (T2DM). Aggregates of Amylin hormone, which is co-secreted with insulin from the pancreatic β-cells, inhibit the activities of insulin and glucagon and cause T2DM. Importance of the conformationally restricted peptides for drug design against T2DM has been invigorated by recent FDA approval of Symlin, which is a large conformationally restricted peptide. However, Symlin still has some issues including solubility, oral bioavailability and cost of preparation. Herein, we introduced a novel strategy for conformationally restricted peptide design adopting a minimalistic approach for cost reduction. We have demonstrated efficient inhibition of amyloid formation of Amylin and its disruption by a novel class of conformationally restricted β-sheet breaker hybrid peptidomimetics (BSBHps). We have inserted β, γ and δ -aminobenzoic acid separately into an amyloidogenic peptide sequence, synthesized α/β, α/γ and α/δ hybrid peptidomimetics, respectively. Interestingly, we observed the aggregation inhibitory efficacy of α/β and α/γ BSBHps, but not of α/δ analogues. They also disrupt existing amyloids into non-toxic forms. Results may be useful for newer drug design against T2DM as well as other amyloidoses and understanding amyloidogenesis.

Cyclodextrin-Based Metal-Organic Nanotube as Fluorescent Probe for Selective Turn-On Detection of Hydrogen Sulfide in Living Cells Based on H2S-Involved Coordination Mechanism

NASA Astrophysics Data System (ADS)

Xin, Xuelian; Wang, Jingxin; Gong, Chuanfang; Xu, Hai; Wang, Rongming; Ji, Shijie; Dong, Hanxiao; Meng, Qingguo; Zhang, Liangliang; Dai, Fangna; Sun, Daofeng

2016-02-01

Hydrogen sulfide (H2S) has been considered as the third biologically gaseous messenger (gasotransmitter) after nitric oxide (NO) and carbon monoxide (CO). Fluorescent detection of H2S in living cells is very important to human health because it has been found that the abnormal levels of H2S in human body can cause Alzheimer’s disease, cancers and diabetes. Herein, we develop a cyclodextrin-based metal-organic nanotube, CD-MONT-2, possessing a {Pb14} metallamacrocycle for efficient detection of H2S. CD-MONT-2‧ (the guest-free form of CD-MONT-2) exhibits turn-on detection of H2S with high selectivity and moderate sensitivity when the material was dissolved in DMSO solution. Significantly, CD-MONT-2‧ can act as a fluorescent turn-on probe for highly selective detection of H2S in living cells. The sensing mechanism in the present work is based on the coordination of H2S as the auxochromic group to the central Pb(II) ion to enhance the fluorescence intensity, which is studied for the first time.

Accelerated generation of oligodendrocyte progenitor cells from human induced pluripotent stem cells by forced expression of Sox10 and Olig2.

PubMed

Li, Pengyan; Li, Mo; Tang, Xihe; Wang, Shuyan; Zhang, Y Alex; Chen, Zhiguo

2016-11-01

Oligodendrocyte progenitor cells (OPCs) hold great promise for treatment of dysmyelinating disorders, such as multiple sclerosis and cerebral palsy. Recent studies on generation of human OPCs mainly use human embryonic stem cells (hESCs) or neural stem cells (NSCs) as starter cell sources for the differentiation process. However, NSCs are restricted in availability and the present method for generation of oligodendrocytes (OLs) from ESCs often requires a lengthy period of time. Here, we demonstrated a protocol to efficiently derive OPCs from human induced pluripotent stem cells (hiPSCs) by forced expression of two transcription factors (2TFs), Sox10 and Olig2. With this method, PDGFRα + OPCs can be obtained in 14 days and O4 + OPCs in 56 days. Furthermore, OPCs may be able to differentiate to mature OLs that could ensheath axons when co-cultured with rat cortical neurons. The results have implications in the development of autologous cell therapies.

Genetic instability caused by loss of MutS homolog 3 in human colorectal cancer

PubMed Central

Haugen, Astrid C.; Goel, Ajay; Yamada, Kanae; Marra, Giancarlo; Nguyen, Thuy-Phuong; Nagasaka, Takeshi; Kanazawa, Shinsaku; Koike, Junichi; Kikuchi, Yoshinori; Zhong, Xiaoling; Arita, Michitsune; Shibuya, Kazutoshi; Oshimura, Mitsuo; Hemmi, Hiromichi; Boland, Clement Richard; Koi, Minoru

2008-01-01

Microsatellite instability (MSI) is a hallmark of mismatch repair deficiency. High levels of MSI at mono- and dinucleotide repeats in colorectal cancer (CRC) are attributed to inactivation of the mismatch repair genes, hMLH1 and hMSH2. CRC with low levels of MSI (MSI-L) exists; however its molecular basis is unclear. There is another type of MSI - “elevated microsatellite alterations at selected tetranucleotide repeats” - (EMAST) where loci containing [AAAG]n or [ATAG]n repeats are unstable. EMAST is frequent in non-colorectal cancers; however the incidence of EMAST and its cause in CRC is not known. Here, we report that MSH3-knock-down or MSH3-deficient cells exhibit the EMAST phenotype and low levels of mutations at dinucleotide repeats. About 60% of 117 sporadic CRC cases exhibit EMAST. All of the cases defined as MSI-H (16 cases) exhibited high levels of EMAST. Among 101 non-MSI-H cases, all 19 cases of MSI-L and 35 of 82 cases of MSS exhibited EMAST. Although non-MSI-H CRC tissues contained MSH3-negative tumor cells ranging from 2-50% of the total tumor cell population, the tissues exhibiting EMAST contained more MSH3-negative cells (average 31.5%) than did the tissues not exhibiting EMAST (8.4%). Taken together, our results support the idea that MSH3-deficiency causes EMAST or EMAST with low levels of MSI at the loci with dinucleotide repeats in CRC. PMID:18922920

The effect of biofiltration on red blood cells 2.3-diphosphoglycerate and pH.

PubMed

Umimoto, K; Hirai, Y; Hayashi, T; Tanaka, H

2000-12-01

To investigate the effect of biofiltration (BF) on the ability of blood to supply oxygen to the peripheral tissues, a 2 week crossover study was conducted with bicarbonate hemodialysis (BcHD) and BF using 5 male patients with diabetic renal failure as subjects. BcHD and BF were performed for 4 h and 3.5 h per session, respectively. Blood gases, the pH of red blood cells (RBC-pH), and 2. 3-diphosphoglycerate in RBC (RBC-2.3DPG) were measured during each treatment. After a 2 week BF treatment, the plasma HCO3- at the beginning of BF was significantly higher than that of BcHD (p < 0.01), and the blood pH improved with an elevated plasma bicarbonate level (p < 0.05). The RBC-pH at the beginning of BF was higher than that of BcHD (p < 0.05) although the RBC-pH at the end of both therapies increased to similar levels. The RBC-2.3DPG during BcHD remained unchanged, but during BF significantly increased (p < 0.05). Metabolic acidosis was significantly improved by BF with its effect reaching to the RBC intracellular level. The improved metabolic acidosis might occur as a result of the increase in RBC-2.3DPG during BF. This increase in RBC-2.3DPG has the effect of reducing the affinity of oxygen for hemoglobin and allows more oxygen to be delivered to the peripheral tissues although the increase in RBC-pH by dialysis restricts the dissociation of oxygen from hemoglobin.

Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells.

PubMed

Shadrin, Ilya Y; Yoon, Woohyun; Li, Liqing; Shepherd, Neal; Bursac, Nenad

2015-07-10

Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca(2+) indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25-40% of hMSCs (from 4 independent donors) acquired periodic Ca(2+) transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca(2+) current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca(2+) channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

Mutations to PB2 and NP proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells.

PubMed

Danzy, Shamika; Studdard, Lydia R; Manicassamy, Balaji; Solorzano, Alicia; Marshall, Nicolle; García-Sastre, Adolfo; Steel, John; Lowen, Anice C

2014-11-01

Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate. Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low-pathogenic, H1N1 duck isolate in primary human respiratory cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Mutations to PB2 and NP Proteins of an Avian Influenza Virus Combine To Confer Efficient Growth in Primary Human Respiratory Cells

PubMed Central

Danzy, Shamika; Studdard, Lydia R.; Manicassamy, Balaji; Solorzano, Alicia; Marshall, Nicolle; García-Sastre, Adolfo; Steel, John

2014-01-01

ABSTRACT Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate. IMPORTANCE Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low-pathogenic, H1N1 duck isolate in primary human respiratory cells. PMID:25210184

Degradation of oxidatively denatured proteins in Escherichia coli.

PubMed

Davies, K J; Lin, S W

1988-01-01

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal growth restriction and the programming of heart growth and cardiac insulin-like growth factor 2 expression in the lamb.

PubMed

Wang, Kimberley C W; Zhang, Lei; McMillen, I Caroline; Botting, Kimberley J; Duffield, Jaime A; Zhang, Song; Suter, Catherine M; Brooks, Doug A; Morrison, Janna L

2011-10-01

Reduced growth in fetal life together with accelerated growth in childhood, results in a ~50% greater risk of coronary heart disease in adult life. It is unclear why changes in patterns of body and heart growth in early life can lead to an increased risk of cardiovascular disease in adulthood. We aimed to investigate the role of the insulin-like growth factors in heart growth in the growth-restricted fetus and lamb. Hearts were collected from control and placentally restricted (PR) fetuses at 137-144 days gestation and from average (ABW) and low (LBW) birth weight lambs at 21 days of age. We quantified cardiac mRNA expression of IGF-1, IGF-2 and their receptors, IGF-1R and IGF-2R, using real-time RT-PCR and protein expression of IGF-1R and IGF-2R using Western blotting. Combined bisulphite restriction analysis was used to assess DNA methylation in the differentially methylated region (DMR) of the IGF-2/H19 locus and of the IGF-2R gene. In PR fetal sheep, IGF-2, IGF-1R and IGF-2R mRNA expression was increased in the heart compared to controls. LBW lambs had a greater left ventricle weight relative to body weight as well as increased IGF-2 and IGF-2R mRNA expression in the heart, when compared to ABW lambs. No changes in the percentage of methylation of the DMRs of IGF-2/H19 or IGF-2R were found between PR and LBW when compared to their respective controls. In conclusion, a programmed increased in cardiac gene expression of IGF-2 and IGF-2R may represent an adaptive response to reduced substrate supply (e.g. glucose and/or oxygen) in order to maintain heart growth and may be the underlying cause for increased ventricular hypertrophy and the associated susceptibility of cardiomyocytes to ischaemic damage later in life.

Homology modeling and docking studies of human Bcl-2L10 protein.

PubMed

Bhargavi, K; Kalyan Chaitanya, P; Ramasree, D; Vasavi, M; Murthy, D K; Uma, V

2010-12-01

Cancer, an unrestrained proliferation of cells, is one of the lead cause of death. Nearly 12.5 million people are diagnosed with cancer worldwide, 7.5 million people die of which 2.5 million cases are from India. Major cause for cancer is restriction of programmed cell death (apoptosis). Multiple signaling pathways regulate apoptosis. Bcl-2 (B - Cell Lymphomas-2) family proteins play a vital role as central regulators of apoptosis. Bcl-2L10, a novel anti-apoptotic protein, blocks apoptosis by mitochondrial dependent mechanism. The present study evaluates the 3D structure of Bcl-2L10 protein using homology modeling and aims to understand plausible functional and binding interactions between Bcl-2L10 with BH3 domain of BAX using protein - protein docking. The docking studies show binding of BH3 domain at Lys 110, Trp-111, Pro-115, Glu-119 and Asp-127 in the groove of BH 1, 2 and 3 domains of Bcl-2L10. Heterodimerization of anti-apoptotic Bcl-2 and BH3 domain of pro-apoptotic Bcl-2 proteins instigates apoptosis. Profound understanding of Bcl-2 pathway may prove useful in identification of future therapeutic targets for cancer.

Time-Dependent Effect of Encapsulating Alginate Hydrogel on Neurogenic Potential

PubMed Central

Razavi, Shahnaz; Khosravizadeh, Zahra; Bahramian, Hamid; Kazemi, Mohammad

2015-01-01

Objective Due to the restricted potential of neural stem cells for regeneration of central nervous system (CNS) after injury, providing an alternative source for neural stem cells is essential. Adipose derived stem cells (ADSCs) are multipotent cells with properties suitable for tissue engineering. In addition, alginate hydrogel is a biocompatible polysaccharide polymer that has been used to encapsulate many types of cells. The aim of this study was to assess the proliferation rate and level of expression of neural markers; NESTIN, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) in encapsulated human ADSCs (hADSCs) 10 and14 days after neural induction. Materials and Methods In this experimental study, ADSCs isolated from human were cultured in neural induction media and seeded into alginate hydrogel. The rate of proliferation and differentiation of encapsulated cells were evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay, immunocytoflourescent and realtime reverse transcriptase polymerase chain reaction (RT-PCR) analyzes 10 and 14 days after induction. Results The rate of proliferation of encapsulated cells was not significantly changed with time passage. The expression of NESTIN and GFAP significantly decreased on day 14 relative to day 10 (P<0.001) but MAP2 expression was increased. Conclusion Alginate hydrogel can promote the neural differentiation of encapsulated hADSCs with time passage. PMID:26199909

Synthesis of P,N-Heterocycles from ω-Amino-H-Phosphinates: Conformationally Restricted α-Amino Acid Analogs

PubMed Central

Queffelec, Clémence; Ribière, Patrice; Montchamp, Jean-Luc

2009-01-01

P,N-Heterocycles (3-hydroxy-1,3-azaphospholane and 3-hydroxy-1,3-azaphosphorinane-3-oxide) are synthesized in moderate yield from readily available ω-amino-H-phosphinates and aldehydes or ketones via an intramolecular Kabachnik-Fields reaction. The products are conformationally restricted phosphinic analogs of α-amino acids. The multi-gram scale syntheses of the H2N(CH2)nPO2H2 phosphinic precursors (n = 1, 2, 3) and some derivatives are also described. PMID:18855477

Effects of 4 weeks of low-load unilateral resistance training, with and without blood flow restriction, on strength, thickness, V wave, and H reflex of the soleus muscle in men.

PubMed

Colomer-Poveda, David; Romero-Arenas, Salvador; Vera-Ibáñez, Antonio; Viñuela-García, Manuel; Márquez, Gonzalo

2017-07-01

To test the effects of 4 weeks of unilateral low-load resistance training (LLRT), with and without blood flow restriction (BFR), on maximal voluntary contraction (MVC), muscle thickness, volitional wave (V wave), and Hoffmann reflex (H reflex) of the soleus muscle. Twenty-two males were randomly distributed into three groups: a control group (CTR; n = 8); a low-load blood flow restriction resistance training group (BFR-LLRT; n = 7), who were an inflatable cuff to occlude blood flow; and a low-load resistance training group without blood flow restriction (LLRT; n = 7). The training consisted of four sets of unilateral isometric LLRT (25% of MVC) three times a week over 4 weeks. MVC increased 33% (P < 0.001) and 22% (P < 0.01) in the trained leg of both BFR-LLRT and LLRT groups, respectively. The soleus thickness increased 9.5% (P < 0.001) and 6.5% (P < 0.01) in the trained leg of both BFR-LLRT and LLRT groups, respectively. However, neither MVC nor thickness changed in either of the legs tested in the CTR group (MVC -1 and -5%, and muscle thickness 1.9 and 1.2%, for the control and trained leg, respectively). Moreover, V wave and H reflex did not change significantly in all the groups studied (V wave /M wave ratio -7.9 and -2.6%, and H max /M max ratio -3.8 and -4%, for the control and trained leg, respectively). Collectively, the present data suggest that in spite of the changes occurring in soleus strength and thickness, 4 weeks of low-load resistance training, with or without BFR, does not cause any change in neural drive or motoneuronal excitability.

Loss of Dishevelleds disrupts planar polarity in ependymal motile cilia and results in hydrocephalus

PubMed Central

Ohata, Shinya; Nakatani, Jin; Herranz-Pérez, Vicente; Cheng, JrGang; Belinson, Haim; Inubushi, Toshiro; Snider, William D.; García-Verdugo, Jose Manuel; Wynshaw-Boris, Anthony; Álvarez-Buylla, Arturo

2014-01-01

SUMMARY Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1−/−;2flox/flox;3+/− mouse, we show that compound genetic ablation of Dvls causes hydrocephalus. In hGFAP-Cre;Dvl1−/−;2flox/flox;3+/− mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1−/−;2flox/flox;3+/− E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent hydrocephalus. PMID:25043421

Zn(2+)-dependence of the synergistic increase in rat thymocyte cell lethality caused by simultaneous application of 4,5-dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) and H2O2.

PubMed

Saitoh, Shohei; Fukunaga, Eri; Ohtani, Hana; Oyama, Yasuo

2015-09-01

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) is an antifouling agent that is an alternative to organotins such as tributyltin (TBT). Because DCOIT decreases catalase activity, it may increase the susceptibility of cells to oxidative stress. We examined the effects of DCOIT on rat thymocytes suffering from oxidative stress induced by H2O2. The simultaneous application of DCOIT and H2O2 induced a synergistic increase in cell lethality that was completely suppressed by chelating intracellular Zn(2+). Intracellular Zn(2+) concentration was increased by DCOIT at concentrations ranging from 0.1 μM to 3 μM. Although the increase in cell lethality produced by DCOIT alone was less than that produced by TBT alone, a synergistic increase was not induced by the combination of TBT and H2O2. Therefore, these results suggest that DCOIT increases vulnerability to oxidative stress and is more cytotoxic than TBT when oxidative stress is induced by H2O2. Copyright © 2014 Elsevier Ltd. All rights reserved.

Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

NASA Technical Reports Server (NTRS)

Parat, Marie-Odile; Stachowicz, Rafal Z.; Fox, Paul L.

2002-01-01

During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

Vinclozolin, a widely used fungizide, enhanced BaP-induced micronucleus formation in human derived hepatoma cells by increasing CYP1A1 expression.

PubMed

Wu, Xin-Jiang; Lu, Wen-qing; Roos, Peter H; Mersch-Sundermann, Volker

2005-10-15

Vinclozolin, a widely used fungicide, can be identified as a residue in numerous vegetable and fruit samples. To get insight in its genetic toxicity, we investigated the genotoxic effect of vinclozolin in the human derived hepatoma cell line HepG2 using the micronucleus (MN) assay. Additionally, to evaluate the co- or anti-mutagenic potency of vinclozolin, we treated HepG2 cells with different concentrations of vinclozolin for 24 h. Subsequently, the cells were exposed to benzo[a]pyrene (BaP) for 1h. Exposure of HepG2 cells to 50-400 microM vinclozolin alone did not cause any induction of micronuclei. However, a pronounced co-mutagenic effect was observed. MN frequencies caused by BaP increased by 30.6%, 52.8% and 65.3% after pretreatment of the cell cultures with 50, 100 and 200 microM vinclozolin, respectively. The highest concentration (400 microM) of vinclozolin tested caused cytotoxicity. Therefore, micronuclei were not considered for that concentration. To clarify the mechanism of cogenotoxicity, we assayed cytochrome P450 1A1 (CYP1A1), which plays a pivotal role in activation of BaP. Cells exposed to vinclozolin led to significant increase of CYP1A1 expression in Western blot. The result suggested that induction of CYP1A1 by vinclozolin account for its enhancing effect on genotoxicity caused by BaP.

A vaccine targeting mutant IDH1 induces antitumour immunity.

PubMed

Schumacher, Theresa; Bunse, Lukas; Pusch, Stefan; Sahm, Felix; Wiestler, Benedikt; Quandt, Jasmin; Menn, Oliver; Osswald, Matthias; Oezen, Iris; Ott, Martina; Keil, Melanie; Balß, Jörg; Rauschenbach, Katharina; Grabowska, Agnieszka K; Vogler, Isabel; Diekmann, Jan; Trautwein, Nico; Eichmüller, Stefan B; Okun, Jürgen; Stevanović, Stefan; Riemer, Angelika B; Sahin, Ugur; Friese, Manuel A; Beckhove, Philipp; von Deimling, Andreas; Wick, Wolfgang; Platten, Michael

2014-08-21

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4(+) T-helper-1 (TH1) responses. CD4(+) TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4(+) T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

Effects of white light-emitting diode (LED) light exposure with different correlated color temperatures (CCTs) on human lens epithelial cells in culture.

PubMed

Xie, Chen; Li, Xiuyi; Tong, Jianping; Gu, Yangshun; Shen, Ye

2014-01-01

Cataract is the major cause for legal blindness in the world. Oxidative stress on the lens epithelial cells (hLECs) is the most important factor in cataract formation. Cumulative light-exposure from widely used light-emitting diodes (LEDs) may pose a potential oxidative threat to the lens epithelium, due to the high-energy blue light component in the white-light emission from diodes. In the interest of perfecting biosafety standards for LED domestic lighting, this study analyzed the photobiological effect of white LED light with different correlated color temperatures (CCTs) on cultured hLECs. The hLECs were cultured and cumulatively exposed to multichromatic white LED light with CCTs of 2954, 5624, and 7378 K. Cell viability of hLECs was measured by Cell Counting Kit-8 (CCK-8) assay. DNA damage was determined by alkaline comet assay. Intracellular reactive oxygen species (ROS) generation, cell cycle, and apoptosis were quantified by flow cytometry. Compared with 2954 and 5624 K LED light, LED light having a CCT of 7378 K caused overproduction of intracellular ROS and severe DNA damage, which triggered G2 /M arrest and apoptosis. These results indicate that white LEDs with a high CCT could cause significant photobiological damage to hLECs. © 2014 The American Society of Photobiology.

Synthesis and biological evaluation of new 4H-pyrano[2,3-b]quinoline derivatives that block acetylcholinesterase and cell calcium signals, and cause neuroprotection against calcium overload and free radicals.

PubMed

Marco-Contelles, José; León, Rafael; López, Manuela G; García, Antonio G; Villarroya, Mercedes

2006-12-01

The synthesis and biological evaluation of ethyl 5-amino-4-(3-pyridyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylates (9-11) is described. We have found that these compounds inhibit AChE with a mild potency, mitigates the [Ca(2+)](c) triggered by high K(+), and cause neuroprotection against Ca(2+) overloading and free radical-induced neuronal death.

Specific destruction of islet transplants in NOD<-->C57BL/6 and NOD<-->C3H/Tif embryo aggregation chimeras irrespective of allelic differences in beta-cell antigens.

PubMed

Leijon, K; Hillörn, V; Bergqvist, I; Holmberg, D

1995-06-01

We have tested the hypothesis that allelic differences in the antigens expressed by the beta-cells of the islets of Langerhans influence the development of insulitis in the non-obese diabetic (NOD) mouse. Islets of Langerhans from NOD, C57BL/6 and C3H/Tif mice were transplanted under the kidney capsule of NOD<-->C57BL/6 and NOD<-->C3H/Tif embryo aggregation (EA) chimeras and the infiltration was scored 5-7 weeks later. Mononuclear cell infiltration of pancreatic islets was observed in 60% of the NOD<-->C57BL/6 and in 55% of the NOD<-->C3H/Tif EA chimeras. All transplanted EA chimeras that developed insulitis also displayed mononuclear cell infiltrates in the transplants, irrespective of the origin of the transplanted islets. In contrast, no infiltration of transplants was detected in EA chimeras scoring negative for insulitis. These results demonstrate that the specific destruction of islet transplants does not require the expression of NOD specific antigens by the islets. Moreover, the beta-cell destruction appears not to be restricted to NOD-MHC. The correlation between insulitis and transplant beta-cell destruction suggests the possibility that the development of insulitis is a prerequisite for transplant specific destruction. MHC restricted destruction may, therefore, precede the beta-cell destruction of transplanted islets. The chimerism among the mononuclear cells infiltrating the islet transplants was found to correlate with the overall haematopoetic chimerism in each of the individual EA chimeras. This observation suggests that NOD bone marrow, as well as non-NOD bone marrow, generates cells contributing to the beta-cell destruction process.

FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jongseok; Shin, Sooan; Teng, C.-H.

2005-09-02

The generation of intense inflammation in the subarachnoid space in response to meningitis-causing bacteria contributes to brain dysfunction and neuronal injury in bacterial meningitis. Microglia, the major immune effector cells in the central nervous system (CNS), become activated by bacterial components to produce proinflammatory immune mediators. In this study, we showed that FimH adhesin, a tip component of type 1 fimbriae of meningitis-causing Escherichia coli K1, activated the murine microglial cell line, BV-2, which resulted in the production of nitric oxide and the release of tumor necrosis factor-{alpha}. Mitogen-activated protein kinases, ERK and p-38, and nuclear factor-{kappa}B were involved inmore » FimH adhesin-mediated microglial activation. These findings suggest that FimH adhesin contributes to the CNS inflammatory response by virtue of activating microglia in E. coli meningitis.« less

ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2*

PubMed Central

Nishitsuji, Hironori; Sugiyama, Ryuichi; Abe, Makoto; Takaku, Hiroshi

2016-01-01

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation. PMID:26694617

MUC1-specific cytotoxic T lymphocytes eradicate tumors when adoptively transferred in vivo.

PubMed

Mukherjee, P; Ginardi, A R; Tinder, T L; Sterner, C J; Gendler, S J

2001-03-01

We have reported previously that MUC1 transgenic mice with spontaneous tumors of the pancreas (designated MET) naturally develop MHC class I-restricted, MUC1-specific CTLs as tumors progress (P. Mukherjee et al., J. Immunol., 165: 3451-3460, 2000). From these MET mice, we have isolated, expanded, and cloned naturally occurring MUC1-specific CTLs in vitro. In this report, we show that the CTL line is predominantly CD8+ T cells and expresses T-cell receptor Vbeta chains 5.1/5.2, 11, 13, and 2 and Valpha chains 2, 8.3, 3.2, and 11.1/11.2. These CTLs recognize several epitopes on the MUC1 tandem repeat with highest affinity to APGSTAPPA. The CTL clone, on the other hand, is 100% CD8+ cells and expresses a single Vbeta chain of 5.1/5.2 and Valpha2. It recognizes only the H-2Db class I-restricted epitope of MUC1, APGSTAPPA. When adoptively transferred, the CTLs were effective in eradicating MUC1-expressing injected tumor cells including mammary gland cells (C57mg) and B16 melanomas. These results suggest that MUC1-specific CTLs are capable of possibly preventing, or at least substantially delaying, MUC1-expressing tumor formation. To our knowledge, this is the first evidence that demonstrates that the naturally occurring MUC1-specific CTLs isolated from one tumor model has antitumor effects on other MUC1-expressing tumors in vivo. Therefore, our data confirm that MUC1 is an important tumor rejection antigen and can serve as a target for immunotherapy.

NDUFA4L2 protects against ischaemia/reperfusion-induced cardiomyocyte apoptosis and mitochondrial dysfunction by inhibiting complex I.

PubMed

Li, Jianhua; Bai, Caiyan; Guo, Junxia; Liang, Wanqian; Long, Jingning

2017-07-01

Myocardial ischaemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as mitochondrial dysfunction. The aims of the present study were to investigate whether NADH dehydrogenase 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2) on myocardial ischaemia-reperfusion (I/R) injury and the underlying molecular mechanism. The hypoxia-reperfusion (H/R) model was established in vitro using H9c2 cells to simulate I/R injury. NDUFA4L2 and complex I expression levels were detected using RT-PCR and western blot. The apoptosis of H9c2 cells was evaluated by flow cytometry and the expression of Bax and Bcl-2 was detected by western blot. The mitochondrial function was assessed by ATP concentration, mPTP opening and cytochrome c (cyto C) expression. Our data indicated that NDUFA4L2 expression was significantly down-regulated in myocardial H/R injury. Overexpression of NDUFA4L2 led to a dramatic prevention of H/R-induced apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl-2. Meanwhile, augmentation of NDUFA4L2 dramatically prevented mitochondrial dysfunction caused by H/R as reflecting in the increased ATP concentration, delayed mPTP opening, as well as down-regulated cyto C expression. Moreover, complex I activation was heightened and negatively regulated by NDUFA4L2. Silencing complex I conspicuously attenuated cell apoptosis and mitochondrial dysfunction. Taken together, our findings demonstrated that NDUFA4L2 protects against H/R injury by preventing myocardium apoptosis and mitochondrial dysfunction via the complex I, and may be a potential therapeutic approach for attenuating myocardial I/R injury. © 2017 John Wiley & Sons Australia, Ltd.

Delayed puberty caused by hyperthyroidism in ram lambs is not a result of suppression in body growth.

PubMed

Chandrasekhar, Y; D'Occhio, M J; Setchell, B P

1986-03-01

Over a period of 8 weeks ram lambs (16 weeks old) were made hyperthyroidal (serum thyroxine approximately equal to 150 ng/ml, compared with control approximately equal to 48 ng/ml) by daily subcutaneous injections of thyroxine or maintained at a constant body weight by restriction of the feed intake. Hyperthyroidal and restricted-intake lambs remained at a constant body weight during the period of treatment whilst control rams gained body weight. Testicular growth was normal in restricted-intake lambs but was suppressed in hyperthyroidal animals. Hyperthyroidism, but not feed restriction, was also associated with decrease in LH pulse frequency (1.3 +/- 0.3/12 h compared with controls 4.8 +/- 0.9/12 h. Hyperthyroidal lambs showed normal LH responses to exogenous LHRH. After cessation of treatment testicular growth continued to be suppressed for up to 16 weeks in previously hyperthyroidic rams; thereafter testes began to increase in size but at 30 weeks after treatment were still smaller than those of control rams. It is concluded that elevated thyroxine concentrations directly influence sexual maturation in ram lambs through actions at hypothalamic and/or higher brain centres which control LH secretion. Transient hyperthyroidism during sexual maturation may cause permanent impairment of sexual development.

High salt-induced excess reactive oxygen species production resulted in heart tube malformation during gastrulation.

PubMed

Gao, Lin-Rui; Wang, Guang; Zhang, Jing; Li, Shuai; Chuai, Manli; Bao, Yongping; Hocher, Berthold; Yang, Xuesong

2018-09-01

An association has been proved between high salt consumption and cardiovascular mortality. In vertebrates, the heart is the first functional organ to be formed. However, it is not clear whether high-salt exposure has an adverse impact on cardiogenesis. Here we report high-salt exposure inhibited basement membrane breakdown by affecting RhoA, thus disturbing the expression of Slug/E-cadherin/N-cadherin/Laminin and interfering with mesoderm formation during the epithelial-mesenchymal transition(EMT). Furthermore, the DiI + cell migration trajectory in vivo and scratch wound assays in vitro indicated that high-salt exposure restricted cell migration of cardiac progenitors, which was caused by the weaker cytoskeleton structure and unaltered corresponding adhesion junctions at HH7. Besides, down-regulation of GATA4/5/6, Nkx2.5, TBX5, and Mef2c and up-regulation of Wnt3a/β-catenin caused aberrant cardiomyocyte differentiation at HH7 and HH10. High-salt exposure also inhibited cell proliferation and promoted apoptosis. Most importantly, our study revealed that excessive reactive oxygen species(ROS)generated by high salt disturbed the expression of cardiac-related genes, detrimentally affecting the above process including EMT, cell migration, differentiation, cell proliferation and apoptosis, which is the major cause of malformation of heart tubes. © 2018 Wiley Periodicals, Inc.

Alterations in expression of imprinted genes from the H19/IGF2 loci in a multigenerational model of intrauterine growth restriction (IUGR).

PubMed

Gonzalez-Rodriguez, Pablo; Cantu, Jessica; O'Neil, Derek; Seferovic, Maxim D; Goodspeed, Danielle M; Suter, Melissa A; Aagaard, Kjersti M

2016-05-01

The H19/IGF2 imprinted loci have attracted recent attention because of their role in cellular differentiation and proliferation, heritable gene regulation, and in utero or early postnatal growth and development. Expression from the imprinted H19/IGF2 locus involves a complex interplay of 3 means of epigenetic regulation: proper establishment of DNA methylation, promoter occupancy of CTCF, and expression of microRNA-675. We have demonstrated previously in a multigenerational rat model of intrauterine growth restriction the epigenetic heritability of adult metabolic syndrome in a F2 generation. We have further demonstrated abrogation of the F2 adult metabolic syndrome phenotype with essential nutrient supplementation of intermediates along the 1-carbon pathway and shown that alterations in the metabolome precede the adult onset of metabolic syndrome. The upstream molecular and epigenomic mediators underlying these observations, however, have yet to be elucidated fully. In the current study, we sought to characterize the impact of the intrauterine growth-restricted lineage and essential nutrient supplementation on both levels and molecular mediators of H19 and IGF2 gene expression in the F2 generation. F2 intrauterine growth-restricted and sham lineages were obtained by exposing P1 (grandmaternal) pregnant dams to bilateral uterine artery ligation or sham surgery at gestational day 19.5. F1 pups were allocated to the essential nutrient supplemented or control diet at postnatal day 21, and bred at 6-7 weeks of age. Hepatic tissues from the resultant F2 offspring at birth and at weaning (day 21) were obtained. Bisulfite modification and sequencing was employed for methylation analysis. H19 and IGF2 expression was measured by quantitative polymerase chain reaction. Promoter occupancy was quantified by the use of chromatin immunoprecipitation, or ChIP, against CTCF insulator proteins. Growth-restricted F2 on control diet demonstrated significant down-regulation in H19 expression compared with sham lineage (0.7831 vs 1.287; P < .05); however, essential nutrient supplementation diet abrogates this difference (4.995 vs 5.100; P > .05). Conversely, Igf2 was up-regulated by essential nutrient supplemented diet on the sham lineage (2.0 fold, P = .01), an effect that was not observed in the growth restricted offspring. A significant differential methylation was observed in the promoter region of region H19 among the intrauterine growth-restricted lineage (18% vs 25%; P < .05) on a control diet, whereas the essential nutrient supplemented diet was alternately associated with hypermethylation in both lineages (sham: 50%; intrauterine growth restriction: 84%, P < .05). Consistent with essential nutrient supplementation impacting the epigenome, a decrease of CTCF promoter occupancy was observed in CTCF4 of the growth restricted lineage (2.45% vs 0.56%; P < .05) on the control diet, an effect that was repressed with essential nutrient supplementation. Heritable growth restriction is associated with changes in H19 gene expression; these changes are reversible with diet supplementation to favorably impact adult metabolic syndrome. Copyright © 2016. Published by Elsevier Inc.

Bacteriostatic action of streptomycin on ribosomally resistant mutants (rpsL) of Salmonella typhimurium.

PubMed Central

Fernández, R O; Antón, D N

1987-01-01

Incubation of streptomycin-resistant (rpsL) mutants of Salmonella typhimurium in alkaline nutrient medium containing streptomycin brought about an inhibition of cell growth that was readily reversed by removing the antibiotic or neutralizing the medium. Growth inhibition was maximal at pH 8.2 and a streptomycin concentration of 800 micrograms/ml. A similar amount of dihydrostreptomycin had a negligible effect, and 10-times-higher concentrations of this antibiotic were required to reproduce the streptomycin action. Addition of streptomycin (400 micrograms/ml) to rpsL cells in alkaline (pH 8.2) nutrient medium caused inhibition of protein and DNA synthesis and also, but to a lower degree, of RNA synthesis. This effect on macromolecular synthesis was not due to ATP deprivation, since ATP content rose after addition of the antibiotic. At pH 8.2, the rate of entrance of streptomycin increased fourfold with respect to the rate at pH 7.0, leading to a large accumulation of streptomycin into rpsL cells. Uptake of the antibiotic was halted by addition of KCN or chloramphenicol. Equal uptake was obtained with 800 micrograms of dihydrostreptomycin or 400 micrograms of streptomycin per ml, yet the former did not affect cell growth at that concentration. It is concluded that high pH stimulates streptomycin and dihydrostreptomycin uptake by rpsL strains but only streptomycin accumulation causes growth inhibition in cells lacking the high-affinity ribosomal site. PMID:2449121

Ectopic expression of SUPERMAN suppresses development of petals and stamens.

PubMed

Yun, Jae-Young; Weigel, Detlef; Lee, Ilha

2002-01-01

The floral regulatory gene SUPERMAN (SUP) encodes a C2H2 type zinc finger protein that is required for maintaining boundaries between floral organs in Arabidopsis. It has been proposed that the main function of SUP is to balance cell proliferation in the third and fourth whorl of developing flowers, thereby maintaining the boundaries between the two whorls. To gain further insight into the function of SUP, we have ectopically expressed SUP using the promoter of APETALA1 (AP1), a gene that is initially expressed throughout floral meristems and later becomes restricted to the first and second whorls. Flowers of AP1::SUP plants have fewer floral organs, consistent with an effect of SUP on cell proliferation. In addition, the AP1::SUP transgene caused the conversion of petals to sepals and suppressed the development of stamens. The expression of the B function homeotic gene APETALA3 (AP3) and its regulator UNUSUAL FLORAL ORGANS (UFO) were delayed and reduced in AP1::SUP flowers. However, SUP does not act merely through UFO, as constitutive expression of UFO did not rescue the defects in petal and stamen development in AP1::SUP flowers. Together, these results suggest that SUP has both indirect and direct effects on the expression of B function homeotic genes.

CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

PubMed Central

Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C.-K.

2015-01-01

In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

Preliminary performance and life evaluations of a 2-kW arcjet

NASA Technical Reports Server (NTRS)

Morren, W. Earl; Curran, Francis M.

1991-01-01

The first results of a program to expand the operational envelope of low-power arcjets to higher specific impulse and power levels are presented. The performance of a kW-class laboratory model arcjet thruster was characterized at three mass flow rates of a 2:1 mixture of hydrogen and nitrogen at power levels ranging from 1.0 to 2.0 kW. This same thruster was then operated for a total of 300 h at a specific impulse and power level of 550 s and 2.0 kW, respectively, in three continuous 100-h sessions. Thruster operation during the three test segments was stable, and no measurable performance degradation was observed during the test series. Substantial cathode erosion was observed during an inspection following the second 100-h test segment. Most notable was the migration of material from the center of the cathode tip to a ring around a large crater. The anode sustained no significant damage during the endurance test segments. Some difficulty was encountered during start-up after disassembly and inspection following the second 100-h test segment, which caused constrictor erosion. This resulted in a reduced flow restriction and arc chamber pressure, which in turn caused a reduction in the arc impedance.

The glossopharyngeal nerve controls epithelial expression of Sprr2a and Krt13 around taste buds in the circumvallate papilla.

PubMed

Miura, Hirohito; Kusakabe, Yuko; Hashido, Kento; Hino, Akihiro; Ooki, Makoto; Harada, Shuitsu

2014-09-19

Tastants reach the tip of taste bud cells through taste pores which are openings in the epithelium. We found Sprr2a is selectively expressed in the upper layer of the epithelium surrounding taste buds in the circumvallate papilla (CV) where the epithelium is organized into taste pores. Sprr2a is a member of a small proline-rich protein family, which is suggested to be involved in the restitution/migration phase of epithelial wound healing. The expression of Sprr2a was restricted to the upper layer and largely segregated with Ptch1 expression that is restricted to the basal side of the epithelium around the taste buds. Denervation resulted in the gradual loss of Sprr2a-expressing cells over 10 days similarly to that of taste bud cells which is in contrast to the rapid loss of Ptch1 expression. We also found that denervation caused an increase of Keratin (Krt)13 expression around taste buds that corresponded with the disappearance of Sprr2a and Ptch1 expression. Taste buds were surrounded by Krt13-negative cells in the CV in control mice. However, at 6 days post-denervation, taste buds were tightly surrounded by Krt13-positive cells. During taste bud development, taste bud cells emerged together with Krt13-negtive cells, and Sprr2a expression was increased along with the progress of taste bud development. These results demonstrate that regional gene expression surrounding taste buds is associated with taste bud formation and controlled by the innervating taste nerve. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

PubMed Central

Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R.; Rommelaere, Jean; Lacroix, Jeannine

2017-01-01

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo. PMID:29039746

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro.

PubMed

Geiss, Carsten; Kis, Zoltán; Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R; Rommelaere, Jean; Dinsart, Christiane; Lacroix, Jeannine

2017-10-17

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

Cellular and humoral cross-immunity against two H3N2v influenza strains in presumably unexposed healthy and HIV-infected subjects.

PubMed

Agrati, Chiara; Castilletti, Concetta; Cimini, Eleonora; Lapa, Daniele; Quartu, Serena; Caglioti, Claudia; Lanini, Simone; Cattoli, Giovanni; Martini, Federico; Ippolito, Giuseppe; Capobianchi, Maria R

2014-01-01

Human cases of infection due to a novel swine-origin variant of influenza A virus subtype H3N2 (H3N2v) have recently been identified in the United States. Pre-existing humoral and cellular immunity has been recognized as one of the key factors in limiting the infection burden of an emerging influenza virus strain, contributing to restrict its circulation and to mitigate clinical presentation. Aim of this study was to assess humoral and cell-mediated cross immune responses to H3N2v in immuno-competent (healthy donors, n = 45) and immuno-compromised hosts (HIV-infected subjects, n = 46) never exposed to H3N2v influenza strain. Humoral response against i) H3N2v (A/H3N2/Ind/08/11), ii) animal vaccine H3N2 strain (A/H3N2/Min/11/10), and iii) pandemic H1N1 virus (A/H1N1/Cal/07/09) was analysed by hemagglutination inhibition assay; cell-mediated response against the same influenza strains was analysed by ELISpot assay. A large proportion of healthy and HIV subjects displayed cross-reacting humoral and cellular immune responses against two H3N2v strains, suggesting the presence of B- and T-cell clones able to recognize epitopes from emerging viral strains in both groups. Specifically, humoral response was lower in HIV subjects than in HD, and a specific age-related pattern of antibody response against different influenza strains was observed both in HD and in HIV. Cellular immune response was similar between HD and HIV groups and no relationship with age was reported. Finally, no correlation between humoral and cellular immune response was observed. Overall, a high prevalence of HD and HIV patients showing cross reactive immunity against two H3N2v strains was observed, with a slightly lower proportion in HIV persons. Other studies focused on HIV subjects at different stages of diseases are needed in order to define how cross immunity can be affected by advanced immunosuppression.

Age-Associated Defects in EphA2 Signaling Impair the Migration of Human Cardiac Progenitor Cells

PubMed Central

Goichberg, Polina; Kannappan, Ramaswamy; Cimini, Maria; Bai, Yingnan; Sanada, Fumihiro; Sorrentino, Andrea; Signore, Sergio; Kajstura, Jan; Rota, Marcello; Anversa, Piero; Leri, Annarosa

2014-01-01

Background Aging negatively impacts on the function of resident human cardiac progenitor cells (hCPCs). Effective regeneration of the injured heart requires mobilization of hCPCs to the sites of damage. In the young heart, signaling by the guidance receptor EphA2 in response to the ephrin A1 ligand promotes hCPC motility and improves cardiac recovery after infarction. Methods and Results We report that old hCPCs are characterized by cell-autonomous inhibition of their migratory ability ex vivo and impaired translocation in vivo in the damaged heart. EphA2 expression was not decreased in old hCPCs; however, the elevated level of reactive oxygen species in aged cells induced post-translational modifications of the EphA2 protein. EphA2 oxidation interfered with ephrin A1-stimulated receptor auto-phosphorylation, activation of Src family kinases, and caveolin-1-mediated internalization of the receptor. Cellular aging altered the EphA2 endocytic route, affecting the maturation of EphA2-containing endosomes and causing premature signal termination. Over-expression of functionally intact EphA2 in old hCPCs corrected the defects in endocytosis and downstream signaling, enhancing cell motility. Based on the ability of phenotypically young hCPCs to respond efficiently to ephrin A1, we developed a novel methodology for the prospective isolation of live hCPCs with preserved migratory capacity and growth reserve. Conclusions Our data demonstrate that the ephrin A1/EphA2 pathway may serve as a target to facilitate trafficking of hCPCs in the senescent myocardium. Importantly, EphA2 receptor function can be implemented for the selection of hCPCs with high therapeutic potential, a clinically relevant strategy that does not require genetic manipulation of stem cells. PMID:24141256

Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

PubMed

Bardella, Chiara; Al-Dalahmah, Osama; Krell, Daniel; Brazauskas, Pijus; Al-Qahtani, Khalid; Tomkova, Marketa; Adam, Julie; Serres, Sébastien; Lockstone, Helen; Freeman-Mills, Luke; Pfeffer, Inga; Sibson, Nicola; Goldin, Robert; Schuster-Böeckler, Benjamin; Pollard, Patrick J; Soga, Tomoyoshi; McCullagh, James S; Schofield, Christopher J; Mulholland, Paul; Ansorge, Olaf; Kriaucionis, Skirmantas; Ratcliffe, Peter J; Szele, Francis G; Tomlinson, Ian

2016-10-10

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1 R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1 R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx.

PubMed

Shabala, Lana; Walker, Emma J; Eklund, Annelie; Randall-Demllo, Sarron; Shabala, Sergey; Guven, Nuri; Cook, Anthony L; Eri, Rajaraman D

2013-10-01

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux 'signatures' during these stress conditions are poorly characterized. We investigated the kinetics of K(+) , Ca(2+) and H(+) ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non-invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K(+) efflux was observed following acute ER stress with peak K(+) efflux being -30·6 and -138·7 nmolm(-2)  s(-1) for 10 and 50 µg ml(-1) , respectively (p < 0·01). TM-dependent Ca(2+) uptake was more prolonged with peak values of 0·85 and 2·68 nmol m(-2)  s(-1) for 10 and 50 µg ml(-1) TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K(+) efflux and Ca(2+) uptake. While K(+) changes were significantly higher at each time point tested, Ca(2+) uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K(+) efflux and Ca(2+) uptake. Functional assays to investigate the effect of inhibiting K(+) efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K(+) , Ca(2+) and H(+) ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases. Copyright © 2012 John Wiley & Sons, Ltd.

Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA.

PubMed Central

Teebor, G W; Frenkel, K; Goldstein, M S

1984-01-01

HeLa cells grown in the presence of [methyl-3H]thymidine contained large amounts of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in their DNA. When the cells were grown in [6-3H]thymidine and their DNA was labeled to the same specific activity, no HMdU was present. When such [6-3H]thymidine-labeled cells were exposed to increasing amounts of gamma-radiation, small but increasing amounts of HMdU were formed in their DNA. This indicates that HMdU can be formed in DNA by two distinct mechanisms. The first is the result of the transmutation of 3H to 3He (beta decay) in the methyl group of thymidine, leading to formation of a carbocation. This short-lived ion reacts with hydroxide ions of water, yielding the hydroxymethyl group. HMdU that is formed by this mechanism is formed at the rate of beta decay of 3H. It appears only in [methyl-3H]thymidine residues and is present in the DNA of both nonirradiated and gamma-irradiated cells. The second mechanism is the result of the radiolysis of water caused by ionizing radiation. The resultant radical species, particularly hydroxyl radicals, may react with many sites on DNA. When the methyl group of thymine is attacked by hydroxyl radicals, the hydroxymethyl group is formed. The formation of HMdU by this mechanism was detected only when [6-3H]thymidine-labeled cells were used, since transmutation of 3H in position 6 of thymine cannot yield HMdU. PMID:6582490

Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA.

PubMed

Teebor, G W; Frenkel, K; Goldstein, M S

1984-01-01

HeLa cells grown in the presence of [methyl-3H]thymidine contained large amounts of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in their DNA. When the cells were grown in [6-3H]thymidine and their DNA was labeled to the same specific activity, no HMdU was present. When such [6-3H]thymidine-labeled cells were exposed to increasing amounts of gamma-radiation, small but increasing amounts of HMdU were formed in their DNA. This indicates that HMdU can be formed in DNA by two distinct mechanisms. The first is the result of the transmutation of 3H to 3He (beta decay) in the methyl group of thymidine, leading to formation of a carbocation. This short-lived ion reacts with hydroxide ions of water, yielding the hydroxymethyl group. HMdU that is formed by this mechanism is formed at the rate of beta decay of 3H. It appears only in [methyl-3H]thymidine residues and is present in the DNA of both nonirradiated and gamma-irradiated cells. The second mechanism is the result of the radiolysis of water caused by ionizing radiation. The resultant radical species, particularly hydroxyl radicals, may react with many sites on DNA. When the methyl group of thymine is attacked by hydroxyl radicals, the hydroxymethyl group is formed. The formation of HMdU by this mechanism was detected only when [6-3H]thymidine-labeled cells were used, since transmutation of 3H in position 6 of thymine cannot yield HMdU.

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

PubMed

Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

2015-10-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M-/- hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M-/- hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M-/- hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. ©AlphaMed Press.

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

PubMed Central

Quan, Yuan; Yan, Qing; Morales, John E.

2015-01-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M−/− hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M−/− hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M−/− hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. PMID:26285657

Cardio-renal and metabolic adaptations during pregnancy in female rats born small: implications for maternal health and second generation fetal growth.

PubMed

Gallo, Linda A; Tran, Melanie; Moritz, Karen M; Mazzuca, Marc Q; Parry, Laura J; Westcott, Kerryn T; Jefferies, Andrew J; Cullen-McEwen, Luise A; Wlodek, Mary E

2012-02-01

Intrauterine growth restriction caused by uteroplacental insufficiency increases risk of cardiovascular and metabolic disease in offspring. Cardio-renal and metabolic responses to pregnancy are critical determinants of immediate and long-term maternal health. However, no studies to date have investigated the renal and metabolic adaptations in growth restricted offspring when they in turn become pregnant. We hypothesised that the physiological challenge of pregnancy in growth restricted females exacerbates disease outcome and compromises next generation fetal growth. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of gestation in WKY rats and F1 female offspring birth and postnatal body weights were recorded. F1 Control and Restricted females were mated at 4 months and blood pressure, renal and metabolic parameters were measured in late pregnancy and F2 fetal and placental weights recorded. Age-matched non-pregnant Control and Restricted F1 females were also studied. F1 Restricted females were born 10-15% lighter than Controls. Basal insulin secretion and pancreatic β-cell mass were reduced in non-pregnant Restricted females but restored in pregnancy. Pregnant Restricted females, however, showed impaired glucose tolerance and compensatory glomerular hypertrophy, with a nephron deficit but normal renal function and blood pressure. F2 fetuses from Restricted mothers exposed to physiological measures during pregnancy were lighter than Controls highlighting additive adverse effects when mothers born small experience stress during pregnancy. Female rats born small exhibit mostly normal cardio-renal adaptations but altered glucose control during late pregnancy making them vulnerable to lifestyle challenges.

Metabolic alterations due to caloric restriction and every other day feeding in normal and growth hormone receptor knockout mice.

PubMed

Westbrook, Reyhan; Bonkowski, Michael S; Arum, Oge; Strader, April D; Bartke, Andrzej

2014-01-01

Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice.

Histone H2A is required for normal centromere function in Saccharomyces cerevisiae

PubMed Central

Pinto, Inés; Winston, Fred

2000-01-01

Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G2–M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere–kinetochore function during chromosome segregation. PMID:10747028

A novel and simple treatment for control of sulfide induced sewer concrete corrosion using free nitrous acid.

PubMed

Sun, Xiaoyan; Jiang, Guangming; Bond, Philip L; Keller, Jurg; Yuan, Zhiguo

2015-03-01

Improved technologies are currently required for mitigating microbially induced concrete corrosion caused by the oxidation of sulfide to sulfuric acid in sewer systems. This study presents a novel strategy for reducing H2S oxidation on concrete surfaces that accommodate an active corrosion biofilm. The strategy aims to reduce biological oxidation of sulfide through treating the corrosion biofilm with free nitrous acid (FNA, i.e. HNO2). Two concrete coupons with active corrosion activity and surface pH of 3.8 ± 0.3 and 2.7 ± 0.2 were sprayed with nitrite. For both coupons, the H2S uptake rates were reduced by 84%-92% 15 days after the nitrite spray. No obvious recovery of the H2S uptake rate was observed during the entire experimental period (up to 12 months after the spray), indicating the long-term effectiveness of the FNA treatment in controlling the activity of the corrosion-causing biofilms. Live/Dead staining tests on the microorganisms on the concrete coupon surfaces demonstrated that viable bacterial cells decreased by > 80% 39 h after the nitrite spray, suggesting that biofilm cells were killed by the treatment. Examination of a corrosion layer within a suspended solution, containing the corrosion-causing biofilms, indicated that biological activity (ATP level and ratio of viable bacterial cells) was severely decreased by the treatment, confirming the bactericidal effect of FNA on the microorganisms in the biofilms. While field trials are still required to verify its effectiveness, it has been demonstrated here that the FNA spray is potentially a very cheap and effective strategy to reduce sewer corrosion. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biological Characterizations of H5Nx Avian Influenza Viruses Embodying Different Neuraminidases

PubMed Central

Yu, Yuandi; Zhang, Zaoyue; Li, Huanan; Wang, Xiuhui; Li, Bo; Ren, Xingxing; Zeng, Zhaoyong; Zhang, Xu; Liu, Shukai; Hu, Pingsheng; Qi, Wenbao; Liao, Ming

2017-01-01

The H5 subtype virus of Highly Pathogenic Avian Influenza Virus has caused huge economic losses to the poultry industry and is a threat to human health. Until 2010, H5N1 subtype virus was the major genotype in China. Since 2011, reassortant H5N2, H5N6, and H5N8 viruses were identified in domestic poultry in China. The clade 2.3.4.4 H5N6 and H5N8 AIV has now spread to most of China. Clade 2.3.4.4 H5N6 virus has caused 17 human deaths. However, the prevalence, pathogenicity, and transmissibility of the distinct NA reassortment with H5 subtypes viruses (H5Nx) is unknown. We constructed five clade 2.3.4.4 reassortant H5Nx viruses that shared the same HA and six internal gene segments. The NA gene segment was replaced with N1, N2, N6, ΔN6 (with an 11 amino acid deletion at the 58th to 68th of NA stalk region), and N8 strains, respectively. The reassortant viruses with distinct NAs of clade 2.3.4.4 H5 subtype had different degrees of fitness. All reassortant H5Nx viruses formed plaques on MDCK cell monolayers, but the ΔH5N6 grew more efficiently in mammalian and avian cells. The reassortant H5Nx viruses were more virulent in mice as compared to the H5N2 virus. The H5N6 and H5N8 reassortant viruses exhibited enhanced pathogenicity and transmissibility in chickens as compared to the H5N1 reassortant virus. We suggest that comprehensive surveillance work should be undertaken to monitor the H5Nx viruses. PMID:28659898

Asymptomatic HLA-A*02:01–Restricted Epitopes from Herpes Simplex Virus Glycoprotein B Preferentially Recall Polyfunctional CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect HLA Transgenic Mice against Ocular Herpes

PubMed Central

Dervillez, Xavier; Qureshi, Huma; Chentoufi, Aziz A.; Khan, Arif A.; Kritzer, Elizabeth; Yu, David C.; Diaz, Oscar R.; Gottimukkala, Chetan; Kalantari, Mina; Villacres, Maria C.; Scarfone, Vanessa M.; McKinney, Denise M.; Sidney, John; Sette, Alessandro; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

2014-01-01

Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b–restricted epitope (gB498–505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1–seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive, HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342–350 and gB561–569. In contrast, in 10 HLA-A*02:01–positive, HSV-1–seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183–191 and gB441–449). ASYMP individuals had a significantly higher proportion of HSV-gB–specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell–dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell–based herpes vaccine. PMID:24101547

Effects of DDT and Triclosan on Tumor-cell Binding Capacity and Cell-Surface Protein Expression of Human Natural Killer Cells

PubMed Central

Hurd-Brown, Tasia; Udoji, Felicia; Martin, Tamara; Whalen, Margaret M.

2012-01-01

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and triclosan (TCS) are organochlorine (OC) compounds that contaminate the environment, are found in human blood, and have been shown to decrease the tumor-cell killing (lytic) function of human natural killer (NK) cells. NK cells defend against tumor cells and virally infected cells. They bind to these targets, utilizing a variety of cell surface proteins. This study examined concentrations of DDT and TCS that decrease lytic function for alteration of NK binding to tumor targets. Levels of either compound that caused loss of binding function were then examined for effects on expression of cell-surface proteins needed for binding. NK cells exposed to 2.5 μM DDT for 24 h (which caused a greater than 55% loss of lytic function) showed a decrease in NK binding function of about 22%, and a decrease in CD16 cell-surface protein of 20%. NK cells exposed to 5 μM TCS for 24 h showed a decrease in ability to bind tumor cells of 37% and a decrease in expression of CD56 of about 34%. This same treatment caused a decrease in lytic function of greater than 87%. These results indicated that only a portion of the loss of NK lytic function seen with exposures to these compounds could be accounted for by loss of binding function. They also showed that loss of binding function is accompanied by a loss cell-surface proteins important in binding function. PMID:22729613

MicroRNA-138 Regulates DNA Damage Response in Small Cell Lung Cancer Cells by Directly Targeting H2AX.

PubMed

Yang, Huan; Luo, Jinwen; Liu, Zhiguang; Zhou, Rui; Luo, Hong

2015-04-01

Lung cancer is the leading cause of cancer death worldwide and small cell lung cancer (SCLC) accounts for a significant proportion of all lung cancer cases. Even so, the underlying mechanism governing SCLC development remains poorly understood and SCLC related cancer death stands high despite decades of intensive investigation. We noted that both miR-138 and H2AX have been implicated in development of various malignancies. Also, there is a recent report showing the role of miR-138 in mediating DNA damage response by targeting H2AX. In light of these data, we sought to characterize the role of miR-138 for SCLC cell growth and cell-cycle progression by regulating H2AX expression. Results showed that miR-138 is significantly down-regulated in SCLC tumor tissues as well as in three SCLC cell lines. After successfully engineering miR-138 overexpression in one of the SCLC cell lines, NCI-H2081, we observed a remarkable reduction of cell growth and a significant inhibition on cell-cycle progression. Moreover, we were able to show that miR-138 potently inhibits H2AX expression, which suggests that H2AX may serve as a downstream executor for miR-138. Consistent with this hypothesis, we found that engineered H2AX knockdown achieves a similar effect as observed for miR-138 overexpression in terms of SCLC growth and cell cycle regulation. We also showed that H2AX overexpression largely abolished miR-138-mediated SCLC cancer cell growth and cell-cycle progression inhibition, which strongly suggests, at least in vitro, that miR-138 potently regulates SCLC development by targeting H2AX. In addition, we found lower miR-138 expression confers SCLC cells with greater DNA damage repair capacity. Finally, we were able to show miR-138 overexpression inhibits DNA damage repair in SCLC cells while miR-138 knockdown further facilitates DNA damage repair in these cells after IR. To date, there has been no study showing the role of miR-138/H2AX machinery in SCLC development. Our results may shed a light to development of new lines of SCLC diagnosis and treatment approaches.

[Cetuximab in combination with icotinib overcomes the acquired resistance caused by EGFR T790M mutation in non-small cell lung cancer].

PubMed

Wang, Meng; Zhang, Lianmin; Zhao, Xiaoliang; Liu, Jun; Chen, Yulong; Wang, Changli

2014-09-01

The aim of this study was to investigate the effects of combination of icotinib and cetuximab on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC, and provide experimental evidence for rational treatment of NSCLC. The effects of these two agents on cell proliferation, apoptosis, and EGFR-dependent signaling were evaluated using 3-(4, 5-dimethylthiazol-2-yl)- 5-diphenyltetrazolium bromide (MTT) assay, annexin V staining, and Western blotting. The expression of molecular markers of tumor proliferation PCNA and Ki-67 protein was further examined by immunohistochemistry, and the expression of EGFR-signaling-related proteins in tissue sections taken from H1975 tumor xenografts was assessed by Western blot assay. Sensitivity to EGFR inhibitors was detected in human H1975 tumor xenograft in nude mice. The in vitro experiment showed that the proliferative ability of H1975 cells was inhibited in a dose-dependent manner, along with the increasing doses of cetuximab and icotinib, and the combination of cetuximab with icotinib resulted in a more pronounced growth inhibition of the H1975 cells. The apoptosis rate of H1975 cells after treatment with 0.5 µmol/L icotinib and 1 µg/ml cetuximab was (22.03 ± 2.41)% and that after treatment with 5 µmol/L icotinib and 10 µg/ml cetuximab was (42.75 ± 2.49)%, both were significantly higher than that after treatment with the same dose of icotinib or cetuximab alone (P < 0.05). The nude mouse experiment showed that the transplanted tumor was growing to (614.5 ± 10.8) mm(3) in the blank control group and to (611.2 ± 8.7) mm(3) at 28 days after icotinib treatment, but (30.8 ± 2.0) mm(3) in the cetuximab treatment group and 0 mm(3) in the cetuximab combined with icotinib group. There was a significantly decreased expression of Ki-67 and PCNA proteins and down-regulation of phosphorylation of EGFR signaling-related proteins in the cetuximab combined with icotinib group. The combination of icotinib with cetuximab can exert synergistic inhibitory effect on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC H1975 cells, interrupts the EGFR-downstream signaling pathway, and enhances the anticancer activity of chemotherapeutic drugs. Our results provide further experimental evidence for the clinical studies of combination of icotinib with cetuximab in the treatment of NSCLC patients associated with secondary drug resistance caused by T790M mutation of EGFR.

Daily Rhythms of Hunger and Satiety in Healthy Men during One Week of Sleep Restriction and Circadian Misalignment.

PubMed

Sargent, Charli; Zhou, Xuan; Matthews, Raymond W; Darwent, David; Roach, Gregory D

2016-01-29

The impact of sleep restriction on the endogenous circadian rhythms of hunger and satiety were examined in 28 healthy young men. Participants were scheduled to 2 × 24-h days of baseline followed by 8 × 28-h days of forced desynchrony during which sleep was either moderately restricted (equivalent to 6 h in bed/24 h; n = 14) or severely restricted (equivalent to 4 h in bed/24 h; n = 14). Self-reported hunger and satisfaction were assessed every 2.5 h during wake periods using visual analogue scales. Participants were served standardised meals and snacks at regular intervals and were not permitted to eat ad libitum. Core body temperature was continuously recorded with rectal thermistors to determine circadian phase. Both hunger and satiety exhibited a marked endogenous circadian rhythm. Hunger was highest, and satiety was lowest, in the biological evening (i.e., ~17:00-21:00 h) whereas hunger was lowest, and satiety was highest in the biological night (i.e., 01:00-05:00 h). The results are consistent with expectations based on previous reports and may explain in some part the decrease in appetite that is commonly reported by individuals who are required to work at night. Interestingly, the endogenous rhythms of hunger and satiety do not appear to be altered by severe--as compared to moderate--sleep restriction.

Endothelial barrier dysfunction caused by LPS correlates with phosphorylation of HSP27 in vivo.

PubMed

Hirano, S; Rees, R S; Yancy, S L; Welsh, M J; Remick, D G; Yamada, T; Hata, J; Gilmont, R R

2004-02-01

Lung edema during sepsis is triggered by formation of gaps between endothelial cells followed by macrophage infiltration. Endothelial gap formation has been proposed to involve changes in the structure of the actin filament cytoskeleton. Heat shock protein 27 (HSP27) is believed to modulate actin filament dynamics or structure, in a manner dependent on its phosphorylation status. We hypothesized that HSP27 may play a role in endothelial gap formation, by affecting actin dependent events in endothelial cells. As there has been no report concerning HSP27 in lung edema in vivo, we examined induction and phosphorylation of HSP27 in lung following LPS injection, as a model of sepsis. In lung, HSP27 mainly localized in capillary endothelial cells of the alveolus, and in smooth muscle cells of pulmonary arteries. HSP27 became significantly more phosphorylated at 3 h after LPS treatment, while the distribution of HSP27 remained unchanged. Pre-treatment with anti-TNFalpha antibody, which has been shown to reduce lung injury, blocked increases in HSP27 phosphorylation at 3 h. HSP27 phosphorylation was also increased in cultured rat pulmonary arterial endothelial cells (RPAEC) by treatment with TNFalpha, LPS, or H2O2. This phosphorylation was blocked by pre-treatment with SB203580, an inhibitor of the upstream kinase, p38 MAP kinase. Increased endothelial permeability caused by H2O2 in vitro was also blocked by SB203580. The amount of actin associated with HSP27 was reduced after treatment with LPS, or H2O2. In summary, HSP27 phosphorylation temporally correlated with LPS induced pathological endothelial cell gap formation in vivo and in a cell culture model system. This is the first report of increased HSP27 phosphorylation associated with pathological lung injury in an animal model of sepsis.

Sailuotong Prevents Hydrogen Peroxide (H2O2)-Induced Injury in EA.hy926 Cells

PubMed Central

Seto, Sai Wang; Chang, Dennis; Ko, Wai Man; Zhou, Xian; Kiat, Hosen; Bensoussan, Alan; Lee, Simon M. Y.; Hoi, Maggie P. M.; Steiner, Genevieve Z.; Liu, Jianxun

2017-01-01

Sailuotong (SLT) is a standardised three-herb formulation consisting of Panax ginseng, Ginkgo biloba, and Crocus sativus designed for the management of vascular dementia. While the latest clinical trials have demonstrated beneficial effects of SLT in vascular dementia, the underlying cellular mechanisms have not been fully explored. The aim of this study was to assess the ability and mechanisms of SLT to act against hydrogen peroxide (H2O2)-induced oxidative damage in cultured human vascular endothelial cells (EAhy926). SLT (1–50 µg/mL) significantly suppressed the H2O2-induced cell death and abolished the H2O2-induced reactive oxygen species (ROS) generation in a concentration-dependent manner. Similarly, H2O2 (0.5 mM; 24 h) caused a ~2-fold increase in lactate dehydrogenase (LDH) release from the EA.hy926 cells which were significantly suppressed by SLT (1–50 µg/mL) in a concentration-dependent manner. Incubation of SLT (50 µg/mL) increased superoxide dismutase (SOD) activity and suppressed the H2O2-enhanced Bax/Bcl-2 ratio and cleaved caspase-3 expression. In conclusion, our results suggest that SLT protects EA.hy916 cells against H2O2-mediated injury via direct reduction of intracellular ROS generation and an increase in SOD activity. These protective effects are closely associated with the inhibition of the apoptotic death cascade via the suppression of caspase-3 activation and reduction of Bax/Bcl-2 ratio, thereby indicating a potential mechanism of action for the clinical effects observed. PMID:28067784

Impacts of maternal dietary protein intake on fetal survival, growth, and development.

PubMed

Herring, Cassandra M; Bazer, Fuller W; Johnson, Gregory A; Wu, Guoyao

2018-03-01

Maternal nutrition during gestation, especially dietary protein intake, is a key determinant in embryonic survival, growth, and development. Low maternal dietary protein intake can cause embryonic losses, intra-uterine growth restriction, and reduced postnatal growth due to a deficiency in specific amino acids that are important for cell metabolism and function. Of note, high maternal dietary protein intake can also result in intra-uterine growth restriction and embryonic death, due to amino acid excesses, as well as the toxicity of ammonia, homocysteine, and H 2 S that are generated from amino acid catabolism. Maternal protein nutrition has a pronounced impact on fetal programming and alters the expression of genes in the fetal genome. As a precursor to the synthesis of molecules (e.g. nitric oxide, polyamines, and creatine) with cell signaling and metabolic functions, L-arginine (Arg) is essential during pregnancy for growth and development of the conceptus. With inadequate maternal dietary protein intake, Arg and other important amino acids are deficient in mother and fetus. Dietary supplementation of Arg during gestation has been effective in improving embryonic survival and development of the conceptus in many species, including humans, pigs, sheep, mice, and rats. Both the balance among amino acids and their quantity are critical for healthy pregnancies and offspring. Impact statement This review aims at: highlighting adverse effects of elevated levels of ammonia in mother or fetus on embryonic/fetal survival, growth, and development; helping nutritionists and practitioners to understand the mechanisms whereby elevated levels of ammonia in mother or fetus results in embryonic/fetal death, growth restriction, and developmental abnormalities; and bringing, into the attention of nutritionists and practitioners, the problems of excess or inadequate dietary intake of protein or amino acids on pregnancy outcomes in animals and humans. The article provides new, effective means to improve embryonic/fetal survival and growth in mammals.

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

PubMed Central

Guessous, Fadila; Marcinkiewicz, Marek; Polanowska-Grabowska, Renata; Kongkhum, Sudawadee; Heatherly, Daniel; Obrig, Tom; Gear, Adrian R. L.

2005-01-01

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1α (SDF-1α) or SDF-1β and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1α at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1α, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS. PMID:16299328

Uniform Li deposition regulated via three-dimensional polyvinyl alcohol nanofiber networks for effective Li metal anodes.

PubMed

Wang, Gang; Xiong, Xunhui; Lin, Zhihua; Zheng, Jie; Fenghua, Zheng; Li, Youpeng; Liu, Yanzhen; Yang, Chenghao; Tang, Yiwei; Liu, Meilin

2018-05-31

Lithium metal anodes are considered to be the most promising anode material for next-generation advanced energy storage devices due to their high reversible capacity and extremely low anode potential. Nevertheless, the formation of dendritic Li, induced by the repeated breaking and repairing of solid electrolyte interphase layers, always causes poor cycling performance and low coulombic efficiency, as well as serious safety problems, which have hindered the practical application of Li anodes for a long time. Herein, we design an electrode by covering a polyvinyl alcohol layer with a three-dimensional nanofiber network structure through an electrospinning technique. The polar functional groups on the surface of the polymer nanofibers can restrict the deposition of Li along the fibers and regulate the deposition of Li uniformly in the voids between the nanofibers. Owing to the structural features of the polymer, the modified Li|Cu electrode displays excellent cycle stability, with a high coulombic efficiency of 98.6% after 200 cycles at a current density of 1 mA cm-2 under a deposition capacity of 1 mA h cm-2, whilst the symmetric cell using the polymer modified Li anode shows stable cycling with a low hysteresis voltage of ∼80 mV over 600 h at a current density of 5 mA cm-2.

Recent Progress Toward Hydrogen Medicine: Potential of Molecular Hydrogen for Preventive and Therapeutic Applications

PubMed Central

Ohta, Shigeo

2011-01-01

Persistent oxidative stress is one of the major causes of most lifestyle-related diseases, cancer and the aging process. Acute oxidative stress directly causes serious damage to tissues. Despite the clinical importance of oxidative damage, antioxidants have been of limited therapeutic success. We have proposed that molecular hydrogen (H2) has potential as a “novel” antioxidant in preventive and therapeutic applications [Ohsawa et al., Nat Med. 2007: 13; 688-94]. H2 has a number of advantages as a potential antioxidant: H2 rapidly diffuses into tissues and cells, and it is mild enough neither to disturb metabolic redox reactions nor to affect reactive oxygen species (ROS) that function in cell signaling, thereby, there should be little adverse effects of consuming H2. There are several methods to ingest or consume H2, including inhaling hydrogen gas, drinking H2-dissolved water (hydrogen water), taking a hydrogen bath, injecting H2-dissolved saline (hydrogen saline), dropping hydrogen saline onto the eye, and increasing the production of intestinal H2 by bacteria. Since the publication of the first H2 paper in Nature Medicine in 2007, the biological effects of H2 have been confirmed by the publication of more than 38 diseases, physiological states and clinical tests in leading biological/medical journals, and several groups have started clinical examinations. Moreover, H2 shows not only effects against oxidative stress, but also various anti-inflammatory and anti-allergic effects. H2 regulates various gene expressions and protein-phosphorylations, though the molecular mechanisms underlying the marked effects of very small amounts of H2 remain elusive. PMID:21736547

Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antunes, Fernanda; Corazzari, Marco; National Institute for Infectious Diseases IRCCS “Lazzaro Spallanzani”

Melanoma is one of leading cause of tumor death worldwide. Anti-cancer strategy includes combination of different chemo-therapeutic agents as well as radiation; however these treatments have limited efficacy and induce significant toxic effects on healthy cells. One of most promising novel therapeutic approach to cancer therapy is the combination of anti-cancer drugs with calorie restriction. Here we investigated the effect Cisplatin (CDDP), one of the most potent chemotherapeutic agent used to treat tumors, in association with fasting in wild type and mutated BRAF{sup V600E} melanoma cell lines. Here we show that nutrient deprivation can consistently enhance the sensitivity of tumormore » cells to cell death induction by CDDP, also of those malignancies particularly resistant to any treatment, such as oncogenic BRAF melanomas. Mechanistic studies revealed that the combined therapy induced cell death is characterized by ROS accumulation and ATF4 in the absence of ER-stress. In addition, we show that autophagy is not involved in the enhanced sensitivity of melanoma cells to combined CDDP/EBSS-induced apoptosis. While, the exposure to 2-DG further enhanced the apoptotic rate observed in SK Mel 28 cells upon treatment with both CDDP and EBSS. - Highlights: • Calorie restriction associated to chemo-therapeutic drugs enhance cell death induction in many resistant malignancies • Cisplatin in association with starvation significantly increases cell death also in those high resistant melanoma cells bearing BRAF mutations • Combined treatment also including 2-DG results in similar cell death levels in both wild type and mutated BRAF cells.« less

[Corrigendum] Long non‑coding RNA H19 promotes cell proliferation and invasion by acting as a ceRNA of miR‑138 and releasing EZH2 in oral squamous cell carcinoma.

PubMed

Hong, Yonglong; He, Haitao; Sui, Wen; Zhang, Jingge; Zhang, Shenfu; Yang, Dajiang

2018-06-01

Following the publication of this article, we realize that the title appeared incorrectly: This appeared in print as "Long non‑coding RNA H1 promotes cell proliferation and invasion by acting as a ceRNA of miR‑138 and releasing EZH2 in oral squamous cell carcinoma", and the corrected title is now featured above ("H1" should have read as "H19"). Note that this error did not have any bearing on the results reported in the paper, or on the conclusions therein. We regret any inconvenience that this mistake has caused. [the original article was published in the International Journal of Oncology 52: 901‑912, 2018; DOI: 10.3892/ijo.2018.4247].

On the causes and consequences of the uncoupler-like effects of quercetin and dehydrosilybin in H9c2 cells

PubMed Central

Mouithys-Mickalad, Ange; Dostal, Zdenek; Serteyn, Didier; Modriansky, Martin

2017-01-01

Quercetin and dehydrosilybin are polyphenols which are known to behave like uncouplers of respiration in isolated mitochondria. Here we investigated whether the effect is conserved in whole cells. Following short term incubation, neither compound uncouples mitochondrial respiration in whole H9c2 cells below 50μM. However, following hypoxia, or long term incubation, leak (state IV with oligomycin) oxygen consumption is increased by quercetin. Both compounds partially protected complex I respiration, but not complex II in H9c2 cells following hypoxia. In a permeabilised H9c2 cell model, the increase in leak respiration caused by quercetin is lowered by increased [ADP] and is increased by adenine nucleotide transporter inhibitor, atractyloside, but not bongkrekic acid. Both quercetin and dehydrosilybin dissipate mitochondrial membrane potential in whole cells. In the case of quercetin, the effect is potentiated post hypoxia. Genetically encoded Ca++ sensors, targeted to the mitochondria, enabled the use of fluorescence microscopy to show that quercetin decreased mitochondrial [Ca++] while dehydrosilybin did not. Likewise, quercetin decreases accumulation of [Ca++] in mitochondria following hypoxia. Fluorescent probes were used to show that both compounds decrease plasma membrane potential and increase cytosolic [Ca++]. We conclude that the uncoupler-like effects of these polyphenols are attenuated in whole cells compared to isolated mitochondria, but downstream effects are nevertheless apparent. Results suggest that the effect of quercetin observed in whole and permeabilised cells may originate in the mitochondria, while the mechanism of action of cardioprotection by dehydrosilybin may be less dependent on mitochondrial uncoupling than originally thought. Rather, protective effects may originate due to interactions at the plasma membrane. PMID:28977033

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome.

PubMed

Sarkar, Koustav; Han, Seong-Su; Wen, Kuo-Kuang; Ochs, Hans D; Dupré, Loïc; Seidman, Michael M; Vyas, Yatin M

2017-12-15

Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. We sought to define how dysfunctional gene transcription is causally linked to the degree of T H cell deficiency and genomic instability in the XLT/WAS clinical spectrum. In human T H 1- or T H 2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in T H 1 cells relative to T H 2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (T H 1 genes) in T H 1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (T H 2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. Transcriptional R-loop imbalance is a novel molecular defect causative in T H 1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging

PubMed Central

Tang, Duozhuang; Tao, Si; Chen, Zhiyang; Koliesnik, Ievgen Oleksandrovich; Calmes, Philip Gerald; Hoerr, Verena; Han, Bing; Gebert, Nadja; Zörnig, Martin; Löffler, Bettina

2016-01-01

Dietary restriction (DR) improves health, delays tissue aging, and elongates survival in flies and worms. However, studies on laboratory mice and nonhuman primates revealed ambiguous effects of DR on lifespan despite improvements in health parameters. In this study, we analyzed consequences of adult-onset DR (24 h to 1 yr) on hematopoietic stem cell (HSC) function. DR ameliorated HSC aging phenotypes, such as the increase in number of HSCs and the skewing toward myeloid-biased HSCs during aging. Furthermore, DR increased HSC quiescence and improved the maintenance of the repopulation capacity of HSCs during aging. In contrast to these beneficial effects, DR strongly impaired HSC differentiation into lymphoid lineages and particularly inhibited the proliferation of lymphoid progenitors, resulting in decreased production of peripheral B lymphocytes and impaired immune function. The study shows that DR-dependent suppression of growth factors and interleukins mediates these divergent effects caused by DR. Supplementation of insulin-like growth factor 1 partially reverted the DR-induced quiescence of HSCs, whereas IL-6/IL-7 substitutions rescued the impairment of B lymphopoiesis exposed to DR. Together, these findings delineate positive and negative effects of long-term DR on HSC functionality involving distinct stress and growth signaling pathways. PMID:26951333

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation.

PubMed

Liangpunsakul, Suthat; Wou, Sung-Eun; Zeng, Yan; Ross, Ruth A; Jayaram, Hiremagalur N; Crabb, David W

2008-12-01

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.

Applicability of Hydrogen Peroxide in Brown Tide Control – Culture and Microcosm Studies

PubMed Central

Randhawa, Varunpreet; Thakkar, Megha; Wei, Liping

2012-01-01

Brown tide algal blooms, caused by the excessive growth of Aureococcus anophagefferens, recur in several northeastern US coastal bays. Direct bloom control could alleviate the ecological and economic damage associated with bloom outbreak. This paper explored the effectiveness and safety of natural chemical biocide hydrogen peroxide (H2O2) for brown tide bloom control. Culture studies showed that H2O2 at 1.6 mg L−1 effectively eradicated high density A. anophagefferens within 24-hr, but caused no significant growth inhibition in the diatoms, prymnesiophytes, green algae and dinoflagellates of >2–3 μm cell sizes among 12 phytoplankton species tested over 1-week observation. When applied to brown tide bloom prone natural seawater in a microcosm study, this treatment effectively removed the developing brown tide bloom, while the rest of phytoplankton assemblage (quantified via HPLC based marker pigment analyses), particularly the diatoms and green algae, experienced only transient suppression then recovered with total chlorophyll a exceeding that in the controls within 72-hr; cyanobacteria was not eradicated but was still reduced about 50% at 72-hr, as compared to the controls. The action of H2O2 against phytoplankton as a function of cell size and cell wall structure, and a realistic scenario of H2O2 application were discussed. PMID:23082223

Stabilization of heavy metals in municipal sewage sludge by freeze-thaw treatment with a blend of diatomite, FeSO4, and Ca(OH)2.

PubMed

Wang, Jing; Fu, Rongbing; Xu, Zhen

2017-08-01

In this work, the effects of diatomite with 15% FeSO 4 •7H 2 O and 7.5% Ca(OH) 2 on sludge stabilization were investigated using batch leaching tests. The influence of cell rupture caused by freezing and thawing on stabilization was also evaluated. The results indicated that the optimal diatomite percentage was 2%. Cell rupture by freezing and thawing reduced heavy metal leachability, followed by cell death and decrease of organic groups. The concentration of heavy metals in sludge leachate increased after cell rupture, indicating that the heavy metal leachability was reduced after freezing and thawings. Moreover, the stabilization effects were generally improved after freezing and thawing. As compared with the stabilization of the original sludge, the unstable fractions decreased and the residual fractions of the heavy metals increased in the stabilized sludge after cell rupture. This study developed a method to stabilize heavy metals in municipal sewage sludge. Diatomite combined with FeSO 4 ·7H 2 O and Ca(OH) 2 improved the treatment of sewage sludge contaminated by heavy metals. Cell lysis by freeze-thaw treatment reduced the risk of leaching heavy metals caused by cell death and decreased major organic groups in the sludge.

Immunology mini-review: the basics of T(H)17 and interleukin-6 in transplantation.

PubMed

Nakagiri, T; Inoue, M; Minami, M; Shintani, Y; Okumura, M

2012-05-01

The outcomes of organ transplantation are determined by graft rejection, the mechanisms of which are some of the most important areas of study in the transplantation field. The main cause of rejection is the immunologic response of the recipient toward the transplanted organ. The immunologic responses are regulated by T-cell subsets, especially helper T-cells, which have been characterized as T(H)1 or T(H)2 cells according to their profiles of cytokines production. A unique subset of recently identified lymphocytes, the regulatory T cells (T(reg)s), seem to play a role in tolerance. The recently identified T(H)17 cells are a subset of effector-helper lymphocytes that specifically secrete interleukin (IL) 17. Interestingly, T(H)17 and T(reg) both develop from naïve T cells on stimulation by transforming growth factor β. The difference is only the existence of IL-6, a proinflammatory cytokine. T(H)17 clears pathogens that are not adequately handled by T(H)1 and T(H)2 elements, as well as contributing to autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory diseases. Autoimmune diseases are caused by reactions to self-antigens. T(H)17 (or IL-17) and IL-6 are also thought to be involved in rejection after organ transplantation. We examined the contributions of T(H)17 and IL-6 in bronchiolitis obliterans (BO), the histologic finding in chronic rejection of lung transplantations. Earlier studies have reported that T(H)17 and IL-6 contribute not only to chronic rejection of lung transplantations, but also to the rejection of other solid organs, e.g., heart, liver, and kidney. In addition, prospective avenues of research on T(H)17 and IL-6 in transplantation have emerged from the perspectives of recent studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Patients With Long-QT Syndrome Caused by Impaired hERG-Encoded Kv11.1 Potassium Channel Have Exaggerated Endocrine Pancreatic and Incretin Function Associated With Reactive Hypoglycemia

PubMed Central

Hyltén-Cavallius, Louise; Iepsen, Eva W.; Wewer Albrechtsen, Nicolai J.; Svendstrup, Mathilde; Lubberding, Anniek F.; Hartmann, Bolette; Jespersen, Thomas; Linneberg, Allan; Christiansen, Michael; Vestergaard, Henrik; Pedersen, Oluf; Holst, Jens J.; Kanters, Jørgen K.

2017-01-01

Background: Loss-of-function mutations in hERG (encoding the Kv11.1 voltage-gated potassium channel) cause long-QT syndrome type 2 (LQT2) because of prolonged cardiac repolarization. However, Kv11.1 is also present in pancreatic α and β cells and intestinal L and K cells, secreting glucagon, insulin, and the incretins glucagon-like peptide-1 (GLP-1) and GIP (glucose-dependent insulinotropic polypeptide), respectively. These hormones are crucial for glucose regulation, and long-QT syndrome may cause disturbed glucose regulation. We measured secretion of these hormones and cardiac repolarization in response to glucose ingestion in LQT2 patients with functional mutations in hERG and matched healthy participants, testing the hypothesis that LQT2 patients have increased incretin and β-cell function and decreased α-cell function, and thus lower glucose levels. Methods: Eleven patients with LQT2 and 22 sex-, age-, and body mass index–matched control participants underwent a 6-hour 75-g oral glucose tolerance test with ECG recording and blood sampling for measurements of glucose, insulin, C-peptide, glucagon, GLP-1, and GIP. Results: In comparison with matched control participants, LQT2 patients had 56% to 78% increased serum insulin, serum C-peptide, plasma GLP-1, and plasma GIP responses (P=0.03–0.001) and decreased plasma glucose levels after glucose ingestion (P=0.02) with more symptoms of hypoglycemia (P=0.04). Sixty-three percent of LQT2 patients developed hypoglycemic plasma glucose levels (<70 mg/dL) versus 36% control participants (P=0.16), and 18% patients developed serious hypoglycemia (<50 mg/dL) versus none of the controls. LQT2 patients had defective glucagon responses to low glucose, P=0.008. β-Cell function (Insulin Secretion Sensitivity Index-2) was 2-fold higher in LQT2 patients than in controls (4398 [95% confidence interval, 2259–8562] versus 2156 [1961–3201], P=0.03). Pharmacological Kv11.1 blockade (dofetilide) in rats had similar effect, and small interfering RNA inhibition of hERG in β and L cells increased insulin and GLP-1 secretion up to 50%. Glucose ingestion caused cardiac repolarization disturbances with increased QTc intervals in both patients and controls, but with a 122% greater increase in QTcF interval in LQT2 patients (P=0.004). Conclusions: Besides a prolonged cardiac repolarization phase, LQT2 patients display increased GLP-1, GIP, and insulin secretion and defective glucagon secretion, causing decreased plasma glucose and thus increased risk of hypoglycemia. Furthermore, glucose ingestion increased QT interval and aggravated the cardiac repolarization disturbances in LQT2 patients. Clinical Trial Registration: URL: http://clinicaltrials.gov. Unique identifier: NCT02775513. PMID:28235848

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift

PubMed Central

Armstrong, Jane A.; Cash, Nicole J.; Ouyang, Yulin; Morton, Jack C.; Chvanov, Michael; Latawiec, Diane; Awais, Muhammad; Tepikin, Alexei V.; Sutton, Robert; Criddle, David N.

2018-01-01

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1–10 μm), whereas higher levels (0.5–1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 μm to 1 mm H2O2), with maximal effects at 500 μm H2O2. H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 μm H2O2. However, higher H2O2 levels (≥50 μm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation. PMID:29626097

[Zn(phen)(O,N,O)(H2O)] and [Zn(phen)(O,N)(H2O)] with O,N,O is 2,6-dipicolinate and N,O is L-threoninate: synthesis, characterization, and biomedical properties.

PubMed

Chin, Lee-Fang; Kong, Siew-Ming; Seng, Hoi-Ling; Tiong, Yee-Lian; Neo, Kian-Eang; Maah, Mohd Jamil; Khoo, Alan Soo-Beng; Ahmad, Munirah; Hor, Tzi-Sum Andy; Lee, Hong-Boon; San, Swee-Lan; Chye, Soi-Moi; Ng, Chew-Hee

2012-10-01

Two ternary Zn(II) complexes, with 1,10-phenanthroline (phen) as the main ligand and a carboxylate-containing ligand [dipicolinate (dipico) or L-threoninate (L-Thr)] as the subsidiary ligand, were prepared and characterized by elemental analysis, Fourier transform IR, UV, and fluorescence spectroscopy, X-ray diffraction, molar conductivity, and electrospray ionization mass spectrometry. X-ray structure analysis shows that both [Zn(phen)(dipico)(H(2)O)]·H(2)O (1) and [Zn(phen)(L-Thr)(H(2)O)Cl]·2H(2)O (2) have octahedral geometry about the Zn(II) atom. Both complexes can inhibit topoisomerase I, and have better anticancer activity than cisplatin against nasopharyngeal cancer cell lines, HK1 and HONE-1, with concentrations causing 50 % inhibition of cell proliferation (IC(50)) in the low micromolar range. Complex 2 has the highest therapeutic index for HK1. Both Zn(II) complexes can induce cell death by apoptosis. Changing the subsidiary ligand in the Zn(II) complexes affects the UV-fluorescence spectral properties of the coordinated phen ligand, the binding affinity for some DNA sequences, nucleobase sequence-selective binding, the phase at which cell cycle progression was arrested for treated cancer cells, and their therapeutic index.

Nonlinear Optical Properties of ZnO for BioimagingCell and Cell Destruction

NASA Astrophysics Data System (ADS)

Urban, Ben; Chakki, Samudyatha; Senthilkumar, Os; Senthilkumar, Kasilingam; Fujita, Yasuhisa; Neogi, Arup

2011-03-01

As of recent years nanotechnology has been at the forefront of scientific research. It promises to have a broad range of applications from turning unhealthy foods into health foods, making computers faster and curing cancer. We present results on using nonlinear optical processes of ZnO nano-crystals to detect, track and destroy cells. By incorporating ZnO into a hydrophobic nano-hydrogel matrix with trace amounts of H2 O2 , we can attach antibodies or microRNA for specific cell targeting and, using the heat generating properties of the third order nonlinear process, release H2 O2 in the cell causing instant cell death. Theoretically, with the appropriate sequence for microRNA or the appropriate antibodies, we could target cancer cells in the body and destroy them. This presentation gives our results until now.

Cuphiin D1, the macrocyclic hydrolyzable tannin induced apoptosis in HL-60 cell line.

PubMed

Wang, C C; Chen, L G; Yang, L L

2000-02-28

Cuphiin D1 (CD1), a new macrocyclic hydrolyzable tannin isolated from Cuphea hyssopifolia, has been shown to exert antitumor activity both in vitro and in vivo. In this study, we explored the mechanism of the CD1-induced antitumor effect on human promyelocytic leukemia (HL-60) cells. The results showed that CD1 induced cytotoxicity in HL-60 cells and the IC50 was 16 microM after 36 h treatment. HL-60 cells treated with CD1 for 36 h decreased the uptake of [3H]-labeled thymidine, uridine and leucine in a dose dependent manner. Electron micrographs demonstrated that HL-60 cells treated with 16 microM CD1 for 36 h exhibited chromatin condensation, indicating the apoptosis occurrence. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G2/M phase, and a concomitant increase of cell population at G1 phase. CD1 also caused DNA fragmentation and inhibited Bcl-2 expression in the HL-60 cells. These results suggest that the inhibition of Bcl-2 expression in HL-60 cell might account for the mechanism of CD1-induced apoptosis.

A Highly Sensitive Chemiluminometric Assay for Real-Time Detection of Biological Hydrogen Peroxide Formation.

PubMed

Zhu, Hong; Jia, Zhenquan; Trush, Michael A; Li, Y Robert

2016-05-01

Hydrogen peroxide (H 2 O 2 ) is a major reactive oxygen species (ROS) produced by various cellular sources, especially mitochondria. At high levels, H 2 O 2 causes oxidative stress, leading to cell injury, whereas at low concentrations, this ROS acts as an important second messenger to participate in cellular redox signaling. Detection and measurement of the levels or rates of production of cellular H 2 O 2 are instrumental in studying the biological effects of this major ROS. While a number of assays have been developed over the past decades for detecting and/or quantifying biological H 2 O 2 formation, none has been shown to be perfect. Perhaps there is no perfect assay for sensitively and accurately quantifying H 2 O 2 as well as other ROS in cells, wherein numerous potential reactants are present to interfere with the reliable measurement of the specific ROS. In this context, each assay has its own advantages and intrinsic limitations. This article describes a highly sensitive assay for real-time detection of H 2 O 2 formation in cultured cells and isolated mitochondria. This assay is based on the luminol/horseradish peroxidase-dependent chemiluminescence that is inhibitable by catalase. The article discusses the usefulness and shortcomings of this chemiluminometric assay in detecting biological H 2 O 2 formation induced by beta-lapachone redox cycling with both cells and isolated mitochondria.

A single, low dose of a cGMP recombinant BCG vaccine elicits protective T cell immunity against the human respiratory syncytial virus infection and prevents lung pathology in mice.

PubMed

Céspedes, Pablo F; Rey-Jurado, Emma; Espinoza, Janyra A; Rivera, Claudia A; Canedo-Marroquín, Gisela; Bueno, Susan M; Kalergis, Alexis M

2017-02-01

Human respiratory syncytial virus (hRSV) is a major health burden worldwide, causing the majority of hospitalizations in children under two years old due to bronchiolitis and pneumonia. HRSV causes year-to-year outbreaks of disease, which also affects the elderly and immunocompromised adults. Furthermore, both hRSV morbidity and epidemics are explained by a consistently high rate of re-infections that take place throughout the patient life. Although significant efforts have been invested worldwide, currently there are no licensed vaccines to prevent hRSV infection. Here, we describe that a recombinant Bacillus Calmette-Guerin (BCG) vaccine expressing the nucleoprotein (N) of hRSV formulated under current good manufacture practices (cGMP rBCG-N-hRSV) confers protective immunity to the virus in mice. Our results show that a single dose of the GMP rBCG-N-hRSV vaccine retains its capacity to protect mice against a challenge with a disease-causing infection of 1×10 7 plaque-forming units (PFUs) of the hRSV A2 clinical strain 13018-8. Compared to unimmunized infected controls, vaccinated mice displayed reduced weight loss and less infiltration of neutrophils within the airways, as well as reduced viral loads in bronchoalveolar lavages, parameters that are characteristic of hRSV infection in mice. Also, ex vivo re-stimulation of splenic T cells at 28days post-immunization activated a repertoire of T cells secreting IFN-γ and IL-17, which further suggest that the rBCG-N-hRSV vaccine induced a mixed, CD8 + and CD4 + T cell response capable of both restraining viral spread and preventing damage of the lungs. All these features support the notion that rBCG-N-hRSV is a promising candidate vaccine to be used in humans to prevent the disease caused by hRSV in the susceptible population. Copyright © 2016 Elsevier Ltd. All rights reserved.

Muscarinic Receptor Activation Protects Cells from Apoptotic Effects of DNA Damage, Oxidative Stress, and Mitochondrial Inhibition*

PubMed Central

De Sarno, Patrizia; Shestopal, Svetlana A.; King, Taj D.; Zmijewska, Anna; Song, Ling; Jope, Richard S.

2006-01-01

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50–200 μm H2O2 caused the activation of caspase-3 beginning after 2–3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H2O2-inducedcaspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H2O2 or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate neurotransmission may exacerbate neuronal loss. PMID:12538580

Formononetin attenuates hydrogen peroxide (H2O2)-induced apoptosis and NF-κB activation in RGC-5 cells.

PubMed

Jia, W-C; Liu, G; Zhang, C-D; Zhang, S-P

2014-01-01

Diabetic retinopathy is a common diabetic eye disease caused by changes in retinal ganglion cells (RGCs). Several studies suggest that the oxidative stress plays a role in the pathogenesis of diabetic retinopathy in adults. Formononetin is a flavone with powerful antioxidant properties that exists naturally in various plants and Chinese medicine. In the present study, an attempt has been made to investigate the antioxidative effects of formononetin on H2O2-induced apoptosis of RGC-5 cells. Exposure of retinal ganglion cells (RGCs) to the indicated concentrations of formononetin and H2O2 for 24 h, analyzed by MTT assay. Cells were stained with Annexin V-FITC and PI, analyzed by flow cytometry. And the level of superoxide anions, malondialdehyde (MDA, a marker of lipid peroxidation), 8-hydroxy-2-deoxyguanosine (8-OHdG, indicator of oxidative DNA damage) and MnSOD (manganese superoxide dismutase) activity were measured by kits. Formononetin reduced hydrogen peroxide (H2O2)-induced apoptosis and improved the levels or activity of indicators of oxidative stress. Formononetin also inhibited the activation of nuclear factor-kappaB (NF-κB), which is a significant transcription factor for RGC-5 apoptosis. Formononetin may be developed as a antioxidant drug to treat diabetic retinopathy.

Type 1 Interferons and NK Cells Limit Murine Cytomegalovirus Escape from the Lymph Node Subcapsular Sinus

PubMed Central

Bruce, Kimberley; Lawler, Clara; Cardin, Rhonda D.

2016-01-01

Cytomegaloviruses (CMVs) establish chronic, systemic infections. Peripheral infection spreads via lymph nodes, which are also a focus of host defence. Thus, this is a point at which systemic infection spread might be restricted. Subcapsular sinus macrophages (SSM) captured murine CMV (MCMV) from the afferent lymph and poorly supported its replication. Blocking the type I interferon (IFN-I) receptor (IFNAR) increased MCMV infection of SSM and of the fibroblastic reticular cells (FRC) lining the subcapsular sinus, and accelerated viral spread to the spleen. Little splenic virus derived from SSM, arguing that they mainly induce an anti-viral state in the otherwise susceptible FRC. NK cells also limited infection, killing infected FRC and causing tissue damage. They acted independently of IFN-I, as IFNAR blockade increased NK cell recruitment, and NK cell depletion increased infection in IFNAR-blocked mice. Thus SSM restricted MCMV infection primarily though IFN-I, with NK cells providing a second line of defence. The capacity of innate immunity to restrict MCMV escape from the subcapsular sinus suggested that enhancing its recruitment might improve infection control. PMID:27926941

Suspension culture process for H9N2 avian influenza virus (strain Re-2).

PubMed

Wang, Honglin; Guo, Suying; Li, Zhenguang; Xu, Xiaoqin; Shao, Zexiang; Song, Guicai

2017-10-01

H9N2 avian influenza virus has caused huge economic loss for the Chinese poultry industry since it was first identified. Vaccination is frequently used as a control method for the disease. Meanwhile suspension culture has become an important tool for the development of influenza vaccines. To optimize the suspension culture conditions for the avian influenza H9N2 virus (Re-2 strain) in Madin-Darby Canine Kidney (MDCK) cells, we studied the culture conditions for cell growth and proliferation parameters for H9N2 virus replication. MDCK cells were successfully cultured in suspension, from a small scale to industrial levels of production, with passage time and initial cell density being optimized. The influence of pH on the culture process in the reactor has been discussed and the process parameters for industrial production were explored via amplification of the 650L reactor. Subsequently, we cultivated cells at high cell density and harvested high amounts of virus, reaching 10log2 (1:1024). Furthermore an animal experiment was conducted to detect antibody. Compared to the chicken embryo virus vaccine, virus cultured from MDCK suspension cells can produce a higher amount of antibodies. The suspension culture process is simple and cost efficient, thus providing a solid foundation for the realization of large-scale avian influenza vaccine production.

Understanding the impact of operational conditions on performance of microbial peroxide producing cells

NASA Astrophysics Data System (ADS)

Young, Michelle N.; Chowdhury, Nadrat; Garver, Emily; Evans, Patrick J.; Popat, Sudeep C.; Rittmann, Bruce E.; Torres, César I.

2017-07-01

Microbial peroxide producing cells (MPPCs) are microbial electrochemical cells used to synthesize hydrogen peroxide (H2O2) in the cathode chamber. Catholyte hydraulic retention time (HRT), different catholytes and their concentrations, and a ferrochelating stabilizer are varied in a continuous-flow cathode MPPC to evaluate their impacts on performance. Using NaCl catholytes, the MPPC produced as high as 3.1 ± 0.37 g H2O2 L-1 at a 4-h HRT with as little as 1.13 W-h g-1 H2O2 energy input and with up to 57 g Lcathode-1 d-1 at a 1-h HRT. For these conditions, the H2O2 production rate provides more than 3 times the H2O2 required for disinfection or micro-pollutant removal while using 5-25% of the power used in conventional H2O2 production processes. Attempts to improve H2O2-production by adding weak acid buffers or H2O2-stabilizing EDTA fail for different reasons. The addition of the ferrochelator EDTA to prevent H2O2 auto-decay deteriorates MPPC performance, because EDTA diffuses from the cathode to the anode, inhibiting iron utilization by anode-respiring bacteria. Weak acid buffers failed to reduce cathode concentration overpotentials. Buffering catholytes lowered the H2O2 yield due to large pH gradients at the cathode chamber entrance, causing the formation of H2O instead of H2O2 or O2 re-formation from H2O2 auto-decay.