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Sample records for nachrs mediate human

  1. Mysterious a6-containing nAChRs: function, pharmacology, and pathophysiology

    Institute of Scientific and Technical Information of China (English)

    Ke-chun YANGU; Guo-zhang JIN; Jie WU

    2009-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are the superfamily of ligand-gated ion channels and widely expressed throughout the central and peripheral nervous systems. nAChRs play crucial roles in modulating a wide range of higher cognitive functions by mediating presynaptic, postsynaptic, and extrasynaptic signaling. Thus far, nine alpha (a2-alO) and three beta ((32, (33, and (34) subunits have been identified in the CNS, and these subunits assemble to form a diversity of functional nAChRs. Although a4(32- and a7-nAChRs are the two major functional nAChR types in the CNS, a6*-nAChRs are abundantly expressed in the midbrain dopaminergic (DAergic) system, including mesocorticolimbic and nigrostriatal pathways, and particularly present in presynaptic nerve terminals. Recently, functional and pharmacological profiles of a6*-nAChRs have been assessed with the use of a6 subunit blockers such as a-conotoxin Mil and PIA, and also by using a6 subunit knockout mice. By modulating DA release in the nucleus accumbens (NAc) and modulating GABA release onto DAergic neurons in the ventral tegmental area (VTA), a6*-nAChRs may play important roles in the mediation of nicotine reward and addiction. Furthermore, a6*-nAChRs in the nigrostriatal DAergic system may be promising targets for selective preventative treatment of Parkinson's disease (PD). Thus, a6*-nAChRs may hold promise for future clinical treatment of human disorders, such as nicotine addiction and PD. In this review, we mainly focus on the recent advances in the understanding of ct6*-nAChR function, pharmacology and pathophysiology.

  2. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Machaalani, R., E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Ghazavi, E. [Bosch Institute, The University of Sydney, NSW 2006 (Australia); School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Hinton, T. [School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Waters, K.A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Hennessy, A. [School of Medicine, University of Western Sydney, NSW 2751 (Australia); Heart Research Institute, 7 Eliza St Newtown, NSW 2042 (Australia)

    2014-05-01

    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

  3. Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.

    Science.gov (United States)

    Henderson, Brandon J; Wall, Teagan R; Henley, Beverley M; Kim, Charlene H; Nichols, Weston A; Moaddel, Ruin; Xiao, Cheng; Lester, Henry A

    2016-03-09

    Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate β2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway.

  4. Age-related changes in functional postsynaptic nAChR subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction

    DEFF Research Database (Denmark)

    Christensen, Mark Holm; Kohlmeier, Kristi Anne

    2016-01-01

    by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within...... that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny...

  5. Modulation of presynaptic nAChRs on postsynaptic GABA receptor in optictectum of juvenile Xenopus

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using the blind patch-clamp technique with the whole-cell mode, we have studied the modulation of presynaptic receptor on postsynaptic γ-aminobutyric acid (GABA) receptor measuring miniature inhibitory postsynaptic currents (mIPSCs) in optic tectum of Xenopus during critical peroid. It was demonstrated that compared with mature neurons, mIPSCs recorded from immature neurons had smaller amplitude and longer decay time. mIPSCs are mediated by GABAa receptor. The nicotinic acetylcholine receptor agonists (carbachol, cytisine, nicotine, DMPP and so on) could increase the frequency of mIPSCs. The enhancement of mIPSCs frequency induced by nAChR agonists was calcium-dependent. However, the choline, a product of hydrolyzed acetylcholine, could not increase the frequency of mIPSCs. DH-β-E, a competitive antagonist of nAChR, blocked the increase of mIPSCs frequency induced by carbachol. Mecamyllamine, an α3β4 subtype of nAChR antagonist, also blocked the carbachol-induced enhancement of mIPSCs. On the other hand, MLA, α7 subtype of nAChR antagonist, had no effect on it. Thus, it seems that nAChR could presynaptically modulate the mIPSCs and α3β4 subtype of nAChR might be involved. But α7 nAChR subtype of nAChR would not be involved. The modulation is calcium- dependent. Meanwhile, we found that Ca2+-free solution could elicit giant PSCs. The frequency of mIPSCs also is related with the level of HP.

  6. nAChR agonist-induced cognition enhancement: integration of cognitive and neuronal mechanisms.

    Science.gov (United States)

    Sarter, Martin; Parikh, Vinay; Howe, William M

    2009-10-01

    The identification and characterization of drugs for the treatment of cognitive disorders has been hampered by the absence of comprehensive hypotheses. Such hypotheses consist of (a) a precisely defined cognitive operation that fundamentally underlies a range of cognitive abilities and capacities and, if impaired, contributes to the manifestation of diverse cognitive symptoms; (b) defined neuronal mechanisms proposed to mediate the cognitive operation of interest; (c) evidence indicating that the putative cognition enhancer facilitates these neuronal mechanisms; (d) and evidence indicating that the cognition enhancer facilitates cognitive performance by modulating these underlying neuronal mechanisms. The evidence on the neuronal and attentional effects of nAChR agonists, specifically agonists selective for alpha4beta2* nAChRs, has begun to support such a hypothesis. nAChR agonists facilitate the detection of signals by augmenting the transient increases in prefrontal cholinergic activity that are necessary for a signal to gain control over behavior in attentional contexts. The prefrontal microcircuitry mediating these effects include alpha4beta2* nAChRs situated on the terminals of thalamic inputs and the glutamatergic stimulation of cholinergic terminals via ionotropic glutamate receptors. Collectively, this evidence forms the basis for hypothesis-guided development and characterization of cognition enhancers.

  7. Effect of calcium on nicotine-induced current expressed by an atypical alpha-bungarotoxin-insensitive nAChR2.

    Science.gov (United States)

    Thany, Steeve H; Courjaret, Raphael; Lapied, Bruno

    2008-06-27

    Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2.

  8. Notion of Mediators in Human Interaction

    OpenAIRE

    Josip Stepanic

    2003-01-01

    Many types of human interaction are mediated processes. However, regarding the details of human description, the mediating unit structure and dynamics is not developed appropriately. The explicit concentration on mediators contributes to understanding of interplay between value sets governing interaction, interaction roles in regular activities and interaction design for purpose. This article is a contribution to development of a concept of mediated human interaction through structuring of me...

  9. Activation of functional α7-containing nAChRs in hippocampal CA1 pyramidal neurons by physiological levels of choline in the presence of PNU-120596.

    Directory of Open Access Journals (Sweden)

    Bopanna I Kalappa

    entire pyramidal neuron and occasionally trigger action potentials. CONCLUSIONS: 1 The majority of hippocampal CA1 pyramidal neurons express functional α7-containing nAChRs. In the absence of PNU-120596, a positive allosteric modulator of α7 nAChRs, a lack of responsiveness of some hippocampal CA1 pyramidal neurons to focal application of 0.5-1 mM choline does not imply a lack of expression of functional α7-containing nAChRs in these neurons. Rather, it may indicate a lack of detection of α7-containing nAChR-mediated currents by patch-clamp electrophysiology. 2 PNU-120596 can serve as a powerful tool for detection and enhancement of responsiveness of low densities of functional α7-containing nAChRs such as those present in hippocampal CA1 pyramidal neurons. 3 In the presence of PNU-120596, physiological concentrations of choline activate functional CA1 pyramidal α7-containing nAChRs and produce step-like currents that cause repetitive step-like depolarizations, occasionally triggering bursts of action potentials in CA1 pyramidal neurons. Therefore, the results of this study suggest that in the presence of PNU-120596 and possibly other positive allosteric modulators, endogenous choline may persistently activate CA1 pyramidal α7-containing nAChRs, enhance the excitability of CA1 pyramidal neurons and thus act as a potent therapeutic agent with potential neuroprotective and cognition-enhancing properties.

  10. Secreted Isoform of Human Lynx1 (SLURP-2)

    DEFF Research Database (Denmark)

    Lyukmanova, E N; Shulepko, M A; Shenkarev, Z O

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we......-evoked currents at concentrations via...... interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs....

  11. The Role of nAChR and Calcium Signaling in Pancreatic Cancer Initiation and Progression

    Energy Technology Data Exchange (ETDEWEB)

    Schaal, Courtney [Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612 (United States); Padmanabhan, Jaya [Department of Molecular Medicine and USF Health Byrd Alzheimer’s Institute, University of South Florida, 4001 E. Fletcher Ave., Tampa, FL 33612 (United States); Chellappan, Srikumar, E-mail: Srikumar.Chellappan@moffitt.org [Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612 (United States)

    2015-07-31

    Pancreatic cancer shows a strong correlation with smoking and the current therapeutic strategies have been relatively ineffective in improving the survival of patients. Efforts have been made over the past many years to understand the molecular events that drive the initiation and progression of pancreatic cancer, especially in the context of smoking. It has become clear that components of tobacco smoke not only initiate these cancers, especially pancreatic ductal adenocarcinomas (PDACs) through their mutagenic properties, but can also promote the growth and metastasis of these tumors by stimulating cell proliferation, angiogenesis, invasion and epithelial-mesenchymal transition. Studies in cell culture systems, animal models and human samples have shown that nicotinic acetylcholine receptor (nAChR) activation enhances these tumor-promoting events by channeling signaling through multiple pathways. In this context, signaling through calcium channels appear to facilitate pancreatic cancer growth by itself or downstream of nAChRs. This review article highlights the role of nAChR downstream signaling events and calcium signaling in the growth, metastasis as well as drug resistance of pancreatic cancer.

  12. Comparative functional expression of nAChR subtypes in rodent DRG neurons

    Directory of Open Access Journals (Sweden)

    Nathan J. Smith

    2013-11-01

    Full Text Available We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: 1 neurons that express predominantly alpha3beta4 and alpha6beta4 nAChRs; 2 neurons that express predominantly alpha7 nAChRs; 3 neurons that express a combination of alpha3beta4/alpha6beta4 and alpha7 nAChRs; and 4 neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels and mitochondrial Ca2+ transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors (mAChRs and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

  13. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    Science.gov (United States)

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-08-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

  14. HUMAN COMMUNICATION AS MEDIATING THE UNITS OF PARAMETERISED ENVIRONMENT

    Directory of Open Access Journals (Sweden)

    Josip Stepanic

    2004-06-01

    Full Text Available Human communication is prevalently a mediated process. Mediators are units of environment, which are attributed functions within the local value set. They are utilised in such a way as to optimise the change of human states. In this article, a mediator-centred interpretation of the human communication is given. The interpretation follows closely the concept of mediated interaction developed within physics. It is conjectured that collection of mediators, which the humans use, has a well-defined average. The averaged collection permits reliable interpretation as a human communication spectrum. Relation of the intensity of a spectral component with regard to different senses, and with regard to intensity of interaction is discussed.

  15. A new IRAK-M-mediated mechanism implicated in the anti-inflammatory effect of nicotine via α7 nicotinic receptors in human macrophages.

    Science.gov (United States)

    Maldifassi, Maria C; Atienza, Gema; Arnalich, Francisco; López-Collazo, Eduardo; Cedillo, Jose L; Martín-Sánchez, Carolina; Bordas, Anna; Renart, Jaime; Montiel, Carmen

    2014-01-01

    Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the 'endotoxin tolerant' phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.

  16. A new IRAK-M-mediated mechanism implicated in the anti-inflammatory effect of nicotine via α7 nicotinic receptors in human macrophages.

    Directory of Open Access Journals (Sweden)

    Maria C Maldifassi

    Full Text Available Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs, has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M, a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3 or two convergent cascades (JAK2/STAT3 and PI3K/STAT3, is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the 'endotoxin tolerant' phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.

  17. Activation of α7nAChR Promotes Diabetic Wound Healing by Suppressing AGE-Induced TNF-α Production.

    Science.gov (United States)

    Dong, Miao-Wu; Li, Ming; Chen, Jie; Fu, Tong-Tong; Lin, Ke-Zhi; Ye, Guang-Hua; Han, Jun-Ge; Feng, Xiang-Ping; Li, Xing-Biao; Yu, Lin-Sheng; Fan, Yan-Yan

    2016-04-01

    Diabetes frequently presents accumulation of advanced glycation end products (AGEs), which might induce excessive TNF-α production from macrophages to cause impaired wound healing. Recent studies have shown that activation of α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages efficiently suppressed TNF-α synthesis. The aim of this study was to investigate the accumulation of AGEs in the wounds and determine whether PNU282987, an α7nAChR agonist, can improve wound repair by inhibiting AGE-mediated TNF-α production in a streptozotocin (STZ)-induced diabetic mouse model. Animals were assigned into four groups: wounded control group, wounded diabetic group, wounded diabetic group treated intraperitoneally with PNU282987, or wounded diabetic group treated intraperitoneally with vehicle. Compared with the non-diabetic control mice, the diabetic mice exhibited delayed wound healing that was characterized by elevated accumulation of AGEs, increased TNF-α level and macrophage infiltration, and decreased fibroblast number and collagen deposition at the late stage of repair. Besides, macrophages of diabetic wounds showed expression of α7nAChR. During late repair, PNU282987 treatment of diabetic mice significantly reduced the level of TNF-α, accelerated wound healing, and elevated fibroblast number and collagen deposition. To investigate the cellular mechanism of these observations, RAW 264.7 cells, a macrophage cell line, were incubated with AGEs in the presence or absence of PNU282987. TNF-α production from AGE-stimulated macrophages was significantly decreased by PNU282987 in a dose-dependent manner. Furthermore, PNU282987 significantly inhibited AGE-induced nuclear factor-κB (NF-κB) activation and receptor for AGE (RAGE) expression. These results strongly suggest that activating α7nAChR can promote diabetic wound healing by suppressing AGE-induced TNF-α production, which may be closely associated with the blockage of NF-κB activation in macrophages.

  18. A novel mode-of-action mediated by the fetal muscle nicotinic acetylcholine receptor resulting in developmental toxicity in rats.

    Science.gov (United States)

    Rasoulpour, Reza J; Ellis-Hutchings, Robert G; Terry, Claire; Millar, Neil S; Zablotny, Carol L; Gibb, Alasdair; Marshall, Valerie; Collins, Toby; Carney, Edward W; Billington, Richard

    2012-06-01

    Sulfoxaflor (X11422208), a novel agricultural molecule, induced fetal effects (forelimb flexure, hindlimb rotation, and bent clavicle) and neonatal death in rats at high doses (≥ 400 ppm in diet); however, no such effects occurred in rabbit dietary studies despite achieving similar maternal and fetal plasma exposure levels. Mode-of-action (MoA) studies were conducted to test the hypothesis that the effects in rats had a single MoA induced by sulfoxaflor agonism on the fetal rat muscle nicotinic acetylcholine receptor (nAChR). The studies included cross-fostering and critical windows of exposure studies in rats, fetal ((α1)(2)β1γδ) and adult ((α1)(2)β1δε) rat and human muscle nAChR in vitro agonism experiments, and neonatal rat phrenic nerve-hemidiaphragm contracture studies. The weight of evidence from these studies supported a novel MoA where sulfoxaflor is an agonist to the fetal, but not adult, rat muscle nAChR and that prolonged agonism on this receptor in fetal/neonatal rats causes sustained striated muscle contracture resulting in concomitant reduction in muscle responsiveness to physiological nerve stimulation. Fetal effects were inducible with as little as 1 day of exposure at the end of gestation, but were rapidly reversible after birth, consistent with a pharmacological MoA. With respect to human relevance, sulfoxaflor was shown to have no agonism on human fetal or adult muscle nAChRs. Taken together, the data support the hypothesis that the developmental effects of sulfoxaflor in rats are mediated via sustained agonism on the fetal muscle nAChR during late fetal development and are considered not relevant to humans.

  19. Human-mediated dispersal of aquatic invertebrates with waterproof footwear

    National Research Council Canada - National Science Library

    Valls, Luis; Castillo-Escrivà, Andreu; Mesquita-Joanes, Francesc; Armengol, Xavier

    2016-01-01

    ...-)national management considering invasive species in other aquatic systems. In this context, we aim at understanding the role of human-mediated dispersal by footwear in protected wetlands with high conservation value...

  20. Human-mediated drivers of change — impacts on coastal ...

    African Journals Online (AJOL)

    Coastal ecosystems are highly vulnerable to human-mediated drivers of global ... wind patterns, ocean current speed and upwelling regimes are all being affected ... exploitation of coastal marine resources, industry (releasing pollutants) and ...

  1. Lipid Mediators and Human Leukemic Blasts

    Directory of Open Access Journals (Sweden)

    Rémi Fiancette

    2011-01-01

    Full Text Available Some of the most potent inflammatory mediators share a lipid origin. They regulate a wide spectrum of cellular processes including cell proliferation and apoptosis. However, the precise roles and ways (if any in which these compounds impact the growth and apoptosis of leukemic blasts remain incompletely resolved. In spite of this, significant advances have been recently made. Here we briefly review the current knowledge about the production of lipid mediators (prostaglandins, leukotrienes, platelet-activating factor by leukemic blasts, the enzymatic activities (phospholipase A2, cyclooxygenases, lipoxygenases involved in their productions and their effects (through specific membrane bound receptors on the growth, and apoptosis of leukemic blasts.

  2. Dog-Mediated Human Rabies Death, Haiti, 2016

    OpenAIRE

    Ryan M Wallace; Etheart, Melissa D.; Doty, Jeff; Monroe, Ben; Crowdis, Kelly; Augustin, Pierre Dilius; Blanton, Jesse; Fenelon, Natael

    2016-01-01

    Haiti has experienced numerous barriers to rabies control over the past decades and is one of the remaining Western Hemisphere countries to report dog-mediated human rabies deaths. We describe the circumstances surrounding a reported human rabies death in 2016 as well as barriers to treatment and surveillance reporting.

  3. Production of inflammatory mediators by human macrophages obtained from ascites

    NARCIS (Netherlands)

    W.M. Pruimboom (Wanda); A.P.J. van Dijk (Arie); C.J.A.M. Tak (Corné); I.L. Bonta; J.H.P. Wilson (Paul); F.J. Zijlstra (Freek)

    1994-01-01

    textabstractAscites is a readily available source of human macrophages (Mø), which can be used to study Mø functions in vitro. We characterized the mediators of inflammation produced by human peritoneal Mø (hp-Mø) obtained from patients with portal hypertension and ascites. The production of the cy

  4. SV40-mediated immortalization of human fibroblasts.

    Science.gov (United States)

    Ozer, H L; Banga, S S; Dasgupta, T; Houghton, J; Hubbard, K; Jha, K K; Kim, S H; Lenahan, M; Pang, Z; Pardinas, J R; Patsalis, P C

    1996-01-01

    We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

  5. Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

    Institute of Scientific and Technical Information of China (English)

    Jian-xin SHEN; Jerrel L YAKEL

    2009-01-01

    Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors (nAChRs), they are grouped into either heteromeric (comprising both α and β subunits) or homomeric (comprising only α subunits) recep-tors. The nAChRs are known to be differentially permeable to calcium ions, with the α7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the α-bungarotoxin-sensitive α7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals: (1) direct calcium influx through the nAChRs, (2) indirect calcium influx through voltage-dependent calcium channels (VDCCs) which are activated by the nAChR-mediated depolarization, and (3) calcium-induced calcium release (CICR) (triggered by the first two sources) from the endoplasmic reticulum (ER) through the ryanodine receptors and inositol (1,4,5)-triphosphate receptors (IP3Rs). Downstream signaling events mediated by nAChR-mediated calcium responses can be grouped into instantaneous effects (such as neurotransmitter release, which can occur in milliseconds after nAChR activation), short-term effects (such as the recovery of nAChR desensitization through cellular signaling cascades), and long-term effects (such as neuroprotection via gene expression). In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understand-ing the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.

  6. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  7. Aquaporins Mediate Silicon Transport in Humans.

    Science.gov (United States)

    Garneau, Alexandre P; Carpentier, Gabriel A; Marcoux, Andrée-Anne; Frenette-Cotton, Rachelle; Simard, Charles F; Rémus-Borel, Wilfried; Caron, Luc; Jacob-Wagner, Mariève; Noël, Micheline; Powell, Jonathan J; Bélanger, Richard; Côté, François; Isenring, Paul

    2015-01-01

    In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1), a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1) the kinetics of substrate transport, 2) their presence in tissues where silicon is presumed to play key roles and 3) their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

  8. Aquaporins Mediate Silicon Transport in Humans.

    Directory of Open Access Journals (Sweden)

    Alexandre P Garneau

    Full Text Available In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1, a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1 the kinetics of substrate transport, 2 their presence in tissues where silicon is presumed to play key roles and 3 their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

  9. Repeated administration of alpha7 nicotinic acetylcholine receptor (nAChR) agonists, but not positive allosteric modulators, increases alpha7 nAChR levels in the brain

    DEFF Research Database (Denmark)

    Christensen, Ditte Z; Mikkelsen, Jens D; Hansen, Henrik H;

    2010-01-01

    -induced phosphorylation of Erk2 in the prefrontal cortex occurs following acute, but not repeated administration. Our results demonstrate that repeated agonist administration increases the number of alpha7 nAChRs in the brain, and leads to coupling versus uncoupling of specific intracellular signaling....... Here we investigate the effects of repeated agonism on alpha7 nAChR receptor levels and responsiveness in vivo in rats. Using [(125)I]-alpha-bungarotoxin (BTX) autoradiography we show that acute or repeated administration with the selective alpha7 nAChR agonist A-582941 increases the number of alpha7 n......-120596 and NS1738 do not increase [(125)I]-BTX binding. Furthermore, A-582941-induced increase in Arc and c-fos mRNA expression in the prefrontal cortex is enhanced and unaltered, respectively, after repeated administration, demonstrating that the alpha7 nAChRs remain responsive. Contrarily, A-582941...

  10. Human milk proresolving mediators stimulate resolution of acute inflammation.

    Science.gov (United States)

    Arnardottir, H; Orr, S K; Dalli, J; Serhan, C N

    2016-05-01

    Human milk contains nutrients and bioactive products relevant to infant development and immunological protection. Here, we investigated the proresolving properties of milk using human milk lipid mediator isolates (HLMIs) and determined their impact on resolution programs in vivo and with human macrophages. HLMIs reduced the maximum neutrophil numbers (14.6±1.2 × 10(6)-11.0±1.0 × 10(6) cells per exudate) and shortened the resolution interval (Ri; 50% neutrophil reduction) by 54% compared with peritonitis. Using rigorous liquid-chromatography tandem-mass spectrometry (LC-MS-MS)-based lipid mediator (LM) metabololipidomics, we demonstrated that human milk possesses a proresolving LM-specialized proresolving mediator (LM-SPM) signature profile, containing SPMs (e.g. resolvins (Rv), protectins (PDs), maresins (MaRs), and lipoxins (LXs)) at bioactive levels (pico-nanomolar concentrations) that enhanced human macrophage efferocytosis and bacterial containment. SPMs identified in human milk included D-series Rvs (e.g., RvD1, RvD2, RvD3, AT-RvD3, and RvD4), PD1, MaR1, E-series Rvs (e.g. RvE1, RvE2, and RvE3), and LXs (LXA4 and LXB4). Of the SPMs identified in human milk, RvD2 and MaR1 (50 ng per mouse) individually shortened Ri by ∼75%. Milk from mastitis gave higher leukotriene B4 and prostanoids and lower SPM levels. Taken together, these findings provide evidence that human milk has proresolving actions via comprehensive LM-SPM profiling, describing a potentially novel mechanism in maternal-infant biochemical imprinting.

  11. Residues Responsible for the Selectivity of α-Conotoxins for Ac-AChBP or nAChRs

    Directory of Open Access Journals (Sweden)

    Bo Lin

    2016-10-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are targets for developing new drugs to treat severe pain, nicotine addiction, Alzheimer disease, epilepsy, etc. α-Conotoxins are biologically and chemically diverse. With 12–19 residues and two disulfides, they can be specifically selected for different nAChRs. Acetylcholine-binding proteins from Aplysia californica (Ac-AChBP are homologous to the ligand-binding domains of nAChRs and pharmacologically similar. X-ray structures of the α-conotoxin in complex with Ac-AChBP in addition to computer modeling have helped to determine the binding site of the important residues of α-conotoxin and its affinity for nAChR subtypes. Here, we present the various α-conotoxin residues that are selective for Ac-AChBP or nAChRs by comparing the structures of α-conotoxins in complex with Ac-AChBP and by modeling α-conotoxins in complex with nAChRs. The knowledge of these binding sites will assist in the discovery and design of more potent and selective α-conotoxins as drug leads.

  12. Carbon nanotubes enhance cytotoxicity mediated by human lymphocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Zhao Sun

    Full Text Available With the expansion of the potential applications of carbon nanotubes (CNT in biomedical fields, the toxicity and biocompatibility of CNT have become issues of growing concern. Since the immune system often mediates tissue damage during pathogenesis, it is important to explore whether CNT can trigger cytotoxicity through affecting the immune functions. In the current study, we evaluated the influence of CNT on the cytotoxicity mediated by human lymphocytes in vitro. The results showed that while CNT at low concentrations (0.001 to 0.1 µg/ml did not cause obvious cell death or apoptosis directly, it enhanced lymphocyte-mediated cytotoxicity against multiple human cell lines. In addition, CNT increased the secretion of IFN-γ and TNF-α by the lymphocytes. CNT also upregulated the NF-κB expression in lymphocytes, and the blockage of the NF-κB pathway reduced the lymphocyte-mediated cytotoxicity triggered by CNT. These results suggest that CNT at lower concentrations may prospectively initiate an indirect cytotoxicity through affecting the function of lymphocytes.

  13. Distribution of bioactive lipid mediators in human skin.

    Science.gov (United States)

    Kendall, Alexandra C; Pilkington, Suzanne M; Massey, Karen A; Sassano, Gary; Rhodes, Lesley E; Nicolaou, Anna

    2015-06-01

    The skin produces bioactive lipids that participate in physiological and pathological states, including homeostasis, induction, propagation, and resolution of inflammation. However, comprehension of the cutaneous lipid complement, and contribution to differing roles of the epidermal and dermal compartments, remains incomplete. We assessed the profiles of eicosanoids, endocannabinoids, N-acyl ethanolamides, and sphingolipids, in human dermis, epidermis, and suction blister fluid. We identified 18 prostanoids, 12 hydroxy-fatty acids, 9 endocannabinoids and N-acyl ethanolamides, and 21 non-hydroxylated ceramides and sphingoid bases, several demonstrating significantly different expression in the tissues assayed. The array of dermal and epidermal fatty acids was reflected in the lipid mediators produced, whereas similarities between lipid profiles in blister fluid and epidermis indicated a primarily epidermal origin of suction blister fluid. Supplementation with omega-3 fatty acids ex vivo showed that their action is mediated through perturbation of existing species and formation of other anti-inflammatory lipids. These findings demonstrate the diversity of lipid mediators involved in maintaining tissue homeostasis in resting skin and hint at their contribution to signaling, cross-support, and functions of different skin compartments. Profiling lipid mediators in biopsies and suction blister fluid can support studies investigating cutaneous inflammatory responses, dietary manipulation, and skin diseases lacking biomarkers and therapeutic targets.

  14. Human-mediated dispersal of seeds over long distances

    Science.gov (United States)

    Wichmann, Matthias C.; Alexander, Matt J.; Soons, Merel B.; Galsworthy, Stephen; Dunne, Laura; Gould, Robert; Fairfax, Christina; Niggemann, Marc; Hails, Rosie S.; Bullock, James M.

    2008-01-01

    Human activities have fundamental impacts on the distribution of species through altered land use, but also directly by dispersal of propagules. Rare long-distance dispersal events have a disproportionate importance for the spread of species including invasions. While it is widely accepted that humans may act as vectors of long-distance dispersal, there are few studies that quantify this process. We studied in detail a mechanism of human-mediated dispersal (HMD). For two plant species we measured, over a wide range of distances, how many seeds are carried by humans on shoes. While over half of the seeds fell off within 5 m, seeds were regularly still attached to shoes after 5 km. Semi-mechanistic models were fitted, and these suggested that long-distance dispersal on shoes is facilitated by decreasing seed detachment probability with distance. Mechanistic modelling showed that the primary vector, wind, was less important as an agent of long-distance dispersal, dispersing seeds less than 250 m. Full dispersal kernels were derived by combining the models for primary dispersal by wind and secondary dispersal by humans. These suggest that walking humans can disperse seeds to very long distances, up to at least 10 km, and provide some of the first quantified dispersal kernels for HMD. PMID:18826932

  15. PXR- and CAR-mediated herbal effect on human diseases.

    Science.gov (United States)

    Xu, Chenshu; Huang, Min; Bi, Huichang

    2016-09-01

    The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are two members of the nuclear receptor superfamily that regulate a broad range of genes involved in drug metabolism and transport. A variety of naturally occurring compounds present in herbal medicines were identified as ligands of PXR and CAR. Recently, accumulative evidences have revealed the PXR- and CAR-mediated herbal effect against multiple human diseases, including inflammatory bowel disease (IBD), cholestatic liver disease, and jaundice. The current review summarized the recent progress in identifying the expanding libraries of herbal medicine as ligands for PXR and CAR. Moreover, the potential for herbal medicines as promising therapeutic agents which were mainly regulated through PXR/CAR signaling pathways was also discussed. The discovery of herbal medicines as modulators of PXR and CAR, and their PXR- and CAR-mediated effect on human diseases will provide a basis for rational drug design, and eventually be explored as a novel therapeutic approach against human diseases. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

  16. Human mast cell mediator cocktail excites neurons in human and guinea-pig enteric nervous system.

    Science.gov (United States)

    Schemann, M; Michel, K; Ceregrzyn, M; Zeller, F; Seidl, S; Bischoff, S C

    2005-04-01

    Neuroimmune interactions are an integral part of gut physiology and involved in the pathogenesis of inflammatory and functional bowel disorders. Mast cells and their mediators are important conveyors in the communication from the innate enteric immune system to the enteric nervous system (ENS). However, it is not known whether a mediator cocktail released from activated human mast cells affects neural activity in the ENS. We used the Multi-Site Optical Recording Technique to image single cell activity in guinea-pig and human ENS after application of a mast cell mediator cocktail (MCMC) that was released from isolated human intestinal mucosa mast cells stimulated by IgE-receptor cross-linking. Local application of MCMC onto individual ganglia evoked an excitatory response consisting of action potential discharge. This excitatory response occurred in 31%, 38% or 11% neurons of guinea-pig submucous plexus, human submucous plexus, or guinea-pig myenteric plexus, respectively. Compound action potentials from nerve fibres or fast excitatory synaptic inputs were not affected by MCMC. This study demonstrates immunoneural signalling in the human gut and revealed for the first time that an MCMC released from stimulated human intestinal mast cells induces excitatory actions in the human and guinea-pig ENS.

  17. Activity of nAChRs containing alpha9 subunits modulates synapse stabilization via bidirectional signaling programs.

    Science.gov (United States)

    Murthy, Vidya; Taranda, Julián; Elgoyhen, A Belén; Vetter, Douglas E

    2009-12-01

    Although the synaptogenic program for cholinergic synapses of the neuromuscular junction is well known, little is known of the identity or dynamic expression patterns of proteins involved in non-neuromuscular nicotinic synapse development. We have previously demonstrated abnormal presynaptic terminal morphology following loss of nicotinic acetylcholine receptor (nAChR) alpha9 subunit expression in adult cochleae. However, the molecular mechanisms underlying these changes have remained obscure. To better understand synapse formation and the role of cholinergic activity in the synaptogenesis of the inner ear, we exploit the nAChR alpha9 subunit null mouse. In this mouse, functional acetylcholine (ACh) neurotransmission to the hair cells is completely silenced. Results demonstrate a premature, effusive innervation to the synaptic pole of the outer hair cells in alpha9 null mice coinciding with delayed expression of cell adhesion proteins during the period of effusive contact. Collapse of the ectopic innervation coincides with an age-related hyperexpression pattern in the null mice. In addition, we document changes in expression of presynaptic vesicle recycling/trafficking machinery in the alpha9 null mice that suggests a bidirectional information flow between the target of the neural innervation (the hair cells) and the presynaptic terminal that is modified by hair cell nAChR activity. Loss of nAChR activity may alter transcriptional activity, as CREB binding protein expression is decreased coincident with the increased expression of N-Cadherin in the adult alpha9 null mice. Finally, by using mice expressing the nondesensitizing alpha9 L9'T point mutant nAChR subunit, we show that increased nAChR activity drives synaptic hyperinnervation.

  18. Phylodynamics and human-mediated dispersal of a zoonotic virus.

    Science.gov (United States)

    Talbi, Chiraz; Lemey, Philippe; Suchard, Marc A; Abdelatif, Elbia; Elharrak, Mehdi; Nourlil, Jalal; Jalal, Nourlil; Faouzi, Abdellah; Echevarría, Juan E; Vazquez Morón, Sonia; Rambaut, Andrew; Campiz, Nicholas; Tatem, Andrew J; Holmes, Edward C; Bourhy, Hervé

    2010-10-28

    Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.

  19. Phylodynamics and human-mediated dispersal of a zoonotic virus.

    Directory of Open Access Journals (Sweden)

    Chiraz Talbi

    Full Text Available Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.

  20. Inflammatory mediators in human epilepsy : A systematic review and meta-analysis

    NARCIS (Netherlands)

    de Vries, Evelien E.; van den Munckhof, Bart; Braun, Kees P J; van Royen-Kerkhof, Annet; de Jager, Wilco; Jansen, Floor E.

    Background: Accumulating evidence suggests a role for inflammation in the pathophysiology of epilepsy. Methods: We performed a systematic review and meta-analysis of studies that investigated inflammatory mediators in human epilepsy. Studies reporting on inflammatory mediators in serum,

  1. Characterization of NAADP-mediated calcium signaling in human spermatozoa

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Tusie, A.A. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Vasudevan, S.R.; Churchill, G.C. [Department of Pharmacology, University of Oxford, Oxford OX1 3QT, England (United Kingdom); Nishigaki, T. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Treviño, C.L., E-mail: ctrevino@ibt.unam.mx [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico)

    2014-01-10

    Highlights: •Human sperm cells synthesize NAADP. •NAADP-AM mediates [Ca{sup 2+}]{sub i} increases in human sperm in the absence of [Ca{sup 2+}]{sub o}. •Human sperm have two acidic compartments located in the head and midpiece. -- Abstract: Ca{sup 2+} signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca{sup 2+}-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca{sup 2+} signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca{sup 2+} and pH. Ca{sup 2+} fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca{sup 2+}] increases in human sperm even in the absence of extracellular Ca{sup 2+}. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-L-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.

  2. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  3. Semisynthetic analogues of toxiferine I and their pharmacological properties at α7 nAChRs, muscle-type nAChRs, and the allosteric binding site of muscarinic M2 receptors

    DEFF Research Database (Denmark)

    Zlotos, D.P.; Tränkle, C; Holzgrabe, U;

    2014-01-01

    , saturation of the exocyclic double bonds, and various N-substituents (methyl, allyl, 4-nitrobenzyl). At the muscle-type nAChRs, most ligands showed similar binding to the muscle relaxant alcuronium, indicating neuromuscular blocking activity, with the nonhydroxylated analogues 3b (Ki = 75 nM) and 3c (Ki = 82...

  4. Repeated administration of alpha7 nicotinic acetylcholine receptor (nAChR) agonists, but not positive allosteric modulators, increases alpha7 nAChR levels in the brain

    DEFF Research Database (Denmark)

    Christensen, Ditte Z; Mikkelsen, Jens D; Hansen, Henrik H;

    2010-01-01

    The alpha7 nicotinic acetylcholine receptor (nAChR) is an important target for treatment of cognitive deficits in schizophrenia and Alzheimer's disease. However, the receptor desensitizes rapidly in vitro, which has led to concern regarding its applicability as a clinically relevant drug target...

  5. Behavioral responses associated with a human-mediated predator shelter.

    Directory of Open Access Journals (Sweden)

    Graeme Shannon

    Full Text Available Human activities in protected areas can affect wildlife populations in a similar manner to predation risk, causing increases in movement and vigilance, shifts in habitat use and changes in group size. Nevertheless, recent evidence indicates that in certain situations ungulate species may actually utilize areas associated with higher levels of human presence as a potential refuge from disturbance-sensitive predators. We now use four-years of behavioral activity budget data collected from pronghorn (Antilocapra americana and elk (Cervus elephus in Grand Teton National Park, USA to test whether predictable patterns of human presence can provide a shelter from predatory risk. Daily behavioral scans were conducted along two parallel sections of road that differed in traffic volume--with the main Teton Park Road experiencing vehicle use that was approximately thirty-fold greater than the River Road. At the busier Teton Park Road, both species of ungulate engaged in higher levels of feeding (27% increase in the proportion of pronghorn feeding and 21% increase for elk, lower levels of alert behavior (18% decrease for pronghorn and 9% decrease for elk and formed smaller groups. These responses are commonly associated with reduced predatory threat. Pronghorn also exhibited a 30% increase in the proportion of individuals moving at the River Road as would be expected under greater exposure to predation risk. Our findings concur with the 'predator shelter hypothesis', suggesting that ungulates in GTNP use human presence as a potential refuge from predation risk, adjusting their behavior accordingly. Human activity has the potential to alter predator-prey interactions and drive trophic-mediated effects that could ultimately impact ecosystem function and biodiversity.

  6. Excipient-mediated supersaturation stabilization in human intestinal fluids.

    Science.gov (United States)

    Bevernage, Jan; Forier, Thomas; Brouwers, Joachim; Tack, Jan; Annaert, Pieter; Augustijns, Patrick

    2011-04-04

    It was the purpose of this study to investigate excipient-mediated precipitation inhibition upon induction of supersaturation of poorly water-soluble drugs in aspirated human intestinal fluids (HIF) representing both the fasted and fed state. Etravirine, ritonavir, loviride, danazol and fenofibrate were selected as model compounds. For comparative purposes, precipitation inhibition was also evaluated in simple aqueous buffer, and in intestinal simulation media representative for the fasted and fed state (FaSSIF and FeSSIF, respectively). Supersaturation was induced in the test media containing predissolved excipient (HPMC-AS, HPMC-E5, HPMC-E50, HPMC-E4M, HPMC-P and PVP) at a defined degree of supersaturation (DS = 20) using the solvent shift method. The results illustrate that cellulosic polymers can reduce the precipitation rate and stabilize supersaturation in HIF. The extent of stabilization was compound and excipient dependent but independent of the nutritional state. Whenever excipient effects were observed, the predictive value of simple buffer or FaSSIF/FeSSIF was rather limited. In general, excipient-mediated precipitation inhibition was less pronounced in HIF compared to simple aqueous buffer or FaSSIF/FeSSIF. However, excipients showing no effect in simple aqueous buffer or FaSSIF/FeSSIF also proved to be ineffective in HIF, indicating the value of these simulation media in the elimination of excipients during formulation development.

  7. Cell mediated immune response in human antirabies revaccination

    Directory of Open Access Journals (Sweden)

    Débora Regina Veiga

    1987-04-01

    Full Text Available The occurrence of secondary cell mediated immune response (CMI in human antirabies immunization was studied. The Puenzalida & Palácios vaccine was used because it is routinely used in Brazil. CMI was evaluated by lymphoblastic transformation indices obtained in whole blood culture in the presence of rabies and control (nervous tissue antigens. Eleven volunteers submitted to revaccination constituted the group under study, while three other volunteers submitted primo vaccination were utilized as control group. A clear secondary CMI to rabies antigen was detected in all the revaccinated volunteers who showed earlier and more intense response than the control group. Response to the control antigen, however, present in all the components of the first group was not detectable in two out of the three primovaccinated and very low in the third one.

  8. Alteration of Cell Cycle Mediated by Zinc in Human Bronchial ...

    Science.gov (United States)

    Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examine cell cycle perturbation upon exposure using a normal human bronchial epithelial cell culture (BEAS-2B). BEAS-2B cells were treated with low (0, 1, 2 µM) and apoptotic (3 µM) doses of Zn2+ plus 1 µM pyrithione, a Zn2+-specific ionophore facilitating cellular uptake, for up to 24 h. Fixed cells were then stained with propidium iodine (PI) and cell cycle phase was determined by fluorescent image cytometry. Initial results report the percentage of cells in the S phase after 18 h exposure to 1, 2, and 3 µM Zn2+ were similar (8%, 7%, and 12%, respectively) compared with 7% in controls. Cells exposed to 3 µM Zn2+ increased cell populations in G2/M phase (76% versus 68% in controls). Interestingly, exposure to 1 µM Zn2+ resulted in decreased (59%) cells in G2/M. While preliminary, these pilot studies suggest Zn2+ alters cell cycle in BEAS-2B cells, particularly in the G2/M phase. The G2/M checkpoint maintains DNA integrity by enabling initiation of DNA repair or apoptosis. Our findings suggest that the adaptive and apoptotic responses to Zn2+ exposure may be mediated via perturbation of the cell cycle at the G2/M checkpoint. This work was a collaborative summer student project. The st

  9. Syntheses and evaluation of halogenated cytisine derivatives and of bioisosteric thiocytisine as potent and selective nAChR ligands.

    Science.gov (United States)

    Imming, P; Klaperski, P; Stubbs, M T; Seitz, G; Gündisch, D

    2001-04-01

    We have developed one-step syntheses of halogenated derivatives of (-)-cytisine featuring a halogen substituent at positions 3, 5 or 3 and 5 of the 2-pyridone fragment, and prepared the novel bioisosteric thiocytisine by oxygen-sulphur exchange. The affinities of these pyridone-modified analogs of (-)-cytisine for (alpha 4)(2)(beta 2)(3) and alpha 7* nAChRs in rat forebrain membranes were determined by competition with (+/-)-[(3)H]epibatidine and [(3)H]MLA, respectively. The 3-halocytisines 7 possess subnanomolar affinities for (alpha 4)(2)(beta 2)(3) nAChRs, higher than those found for (-)-cytisine as well as for the 5-halocytisines 8 and 3,5-dihalocytisines 6. In contrast to the parent alkaloid the 3-halogenated species display much a higher affinity for the alpha 7* nAChR subtype. The most potent molecule was 3-bromocytisine (7b) with preferential selectivity (200-fold) for the (alpha 4)(2)(beta 2)(3) subtype [K(i)=10 pM (alpha 4 beta 2) and 2.0 nM (alpha 7*)]. Replacement of the lactam with a thiolactam pharmacophore to thiocytisine (12) resulted in a subnanomolar affinity for the (alpha 4)(2)(beta 2)(3) nAChR subtype (K(i)=0.832 nM), but in a drastic decrease of affinity for the alpha 7* subtype; thiocytisine (12) has a K(i) value of 4000 nM (alpha 7*), giving a selectivity of 4800-fold for the neuronal (alpha 4)(2)(beta 2)(3)-nAChR and thus displaying the best affinity-selectivity profile in the series under consideration.

  10. PDGF mediates derivation of human embryonic germ cells.

    Science.gov (United States)

    Li, Yang; Hong, Wan Xing; Lan, Baojin; Xu, Xiaoyan; Liu, Yinan; Kong, Lin; Li, Yaxuan; Zhou, Shixin; Liu, Ying; Feng, Ruopeng; Jiang, Sibo; He, Qihua; Tan, Jichun

    2013-01-01

    Human embryonic germ cells (hEGCs) are a valuable and underutilized source of pluripotent stem cells. Unlike embryonic stem cells, which have been extensively studied, little is known about the factors that regulate hEGC derivation and maintenance. This study demonstrates for the first time a central role for selective activation of PDGFR signaling in the derivation and maintenance of pluripotency in hEGCs. In the study, hEGCs were found to express PDGF receptor α at high levels compared to human embryonic stem cells (hESCs). PDGF significantly improved formation of alkaline phosphatase (AP) positive hEGC colonies. We subsequently determined that PDGF activates the phosphatidylinositol-3-kinase (PI3K) pathway as phosphorylation of AKT was up-regulated in response to PDGF. Furthermore, inhibition of PI3K signaling using small molecular inhibitor LY294002 led to significantly decreased AP positive hEGC colony formation whereas inhibition of MAPK pathway using U0126 had a negligible effect. We established a primary mechanism for PDGF mediated derivation and maintenance of hEGCs by demonstrating that OCT4 was upregulated and PTEN was suppressed in a dose dependent manner in response to PDGF.

  11. Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice.

    Science.gov (United States)

    Li, Xiangming; Huang, Jing; Zhang, Min; Funakoshi, Ryota; Sheetij, Dutta; Spaccapelo, Roberta; Crisanti, Andrea; Nussenzweig, Victor; Nussenzweig, Ruth S; Tsuji, Moriya

    2016-08-31

    A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.

  12. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    Science.gov (United States)

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  13. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties.

    Directory of Open Access Journals (Sweden)

    Morten Skøtt Thomsen

    Full Text Available The existence of α7β2 nicotinic acetylcholine receptors (nAChRs has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.

  14. Enantiopure Cyclopropane-Bearing Pyridyldiazabicyclo[3.3.0]octanes as Selective α4β2-nAChR Ligands.

    Science.gov (United States)

    Onajole, Oluseye K; Eaton, J Brek; Lukas, Ronald J; Brunner, Dani; Thiede, Lucinda; Caldarone, Barbara J; Kozikowski, Alan P

    2014-11-13

    We report the synthesis and characterization of a series of enantiopure 5-cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes that display low nanomolar binding affinities and act as functional agonists at α4β2-nicotinic acetylcholine receptor (nAChR) subtype. Structure-activity relationship studies revealed that incorporation of a cyclopropane-containing side chain at the 5-position of the pyridine ring provides ligands with improved subtype selectivity for nAChR β2 subunit-containing nAChR subtypes (β2*-nAChRs) over β4*-nAChRs compared to the parent compound 4. Compound 15 exhibited subnanomolar binding affinity for α4β2- and α4β2*-nAChRs with negligible interaction. Functional assays confirm selectivity for α4β2-nAChRs. Furthermore, using the SmartCube assay system, this ligand showed antidepressant, anxiolytic, and antipsychotic features, while mouse forced-swim assay further confirm the antidepressant-like property of 15.

  15. Bystander apoptosis in human cells mediated by irradiated blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

    2012-03-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

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

    Science.gov (United States)

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

    1998-04-02

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

  17. Neuronal nicotinic acetylcholine receptors serve as sensitive targets that mediate β-amyloid neurotoxicity

    Institute of Scientific and Technical Information of China (English)

    Qiang LIU; Jie WU

    2006-01-01

    Alzheimer's disease (AD) is the most common form of brain dementia characterized by the accumulation of β-amyloid peptides (Aβ) and loss of forebrain cholinergic neurons. Aβ accumulation and aggregation are thought to contribute to cholinergic neuronal degeneration, in turn causing learning and memory deficits, but the specific targets that mediate Aβ neurotoxicity remain elusive. Recently, accumlating lines of evidence have demonstrated that Aβ directly modulates the function of neuronal nicotinic acetylcholine receptors (nAChRs), which leads to the new hypothesis that neuronal nAChRs may serve as important targets that mediate Aβ neurotoxicity. In this review, we summarize current studies performed in our laboratory and in others to address the question of how Aβ modulates neuronal nAChRs, especially nAChR subunit function.

  18. Functionality and stability data of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology

    Directory of Open Access Journals (Sweden)

    Luis F. Padilla-Morales

    2016-03-01

    Full Text Available The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR rich membrane solubilized with long chain (16 saturated carbons lysophospholipid with glycerol headgroup (LFG-16. The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique.

  19. Methylene blue mediated photobiomodulation on human osteoblast cells.

    Science.gov (United States)

    Ateş, Gamze Bölükbaşı; Ak, Ayşe; Garipcan, Bora; Gülsoy, Murat

    2017-08-04

    Photobiomodulation (PBM) and photodynamic therapy (PDT) are two major methods, which use light in medicine and dentistry. PBM uses low-level laser light to induce cell proliferation and activity. In contrast, PDT use laser light combined with a photosensitizer (PS) to cause cell death. Due to similar, not fully understood mechanisms and biphasic response of light, unexpected and complex outcomes may be observed. In the present study, the effect of 635 nm laser light, with power density 50 mW/cm(2), at three different energy densities (0.5, 1, and 2 J/cm(2) which last 10, 20, and 40 s, respectively) mediated by methylene blue (MB) on the human osteoblast cell line (ATCC-CRL-11372, Rockville, MD, USA) was investigated. Cell viability (MTT assay and acridine orange/propidium iodide staining) and proliferation (Alamar Blue assay) were assessed at 24, 48, and 72 h post irradiation. Alkaline phosphatase (ALP) activity, mineralization (Alizarin Red staining) and gene expressions (RT-PCR analysis) were analyzed at 7th and 14th days after treatment. Five groups were formed as the control group (no MB, no irradiation), MB (only 0.05 μM MB), MB + 0.5 J/cm(2), MB + 1 J/cm(2), and MB + 2 J/cm(2). Cell viability was decreased at 72 h (ANOVA; p < 0.05) for MB + 0.5 J/cm(2), MB + 1 J/cm(2), and MB + 2 J/cm(2) groups. Although proliferation does not seem to be effected by MB-mediated laser application, osteo-anabolic activity is altered. ALP activity was significantly increased at day 7 (ANOVA; p < 0.05) for MB-combined laser groups; on the other hand, mineralization was significantly decreased (ANOVA; p < 0.05) in all treatment groups. Alkaline phosphatase and collagen-I expressions were upregulated in MB + 2 J/cm(2) group at 7th and 14th days, respectively. These results may contribute to the low-dose PDT researches and understanding PBM effects on osteoblast behavior but further studies are needed since inappropriate conditions may lead to

  20. Cytotoxicity mediated by human Fc receptors for IgG.

    Science.gov (United States)

    Fanger, M W; Shen, L; Graziano, R F; Guyre, P M

    1989-03-01

    The Fc receptors for IgG(Fc gamma R) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of Fc gamma R-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these Fc gamma R in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monoclonal antibodies specific for Fc gamma R on human leukocytes has established the existence of three distinct Fc gamma R and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of Fc gamma R-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different Fc gamma R is discussed. The results of these studies suggest that the ability of a given Fc gamma R to trigger killing is sometimes dependent on the type of Fc gamma R, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.

  1. Contribution of α4β2 nAChR in nicotine-induced intracellular calcium response and excitability of MSDB neurons.

    Science.gov (United States)

    Wang, Jiangang; Wang, Yali; Wang, Yang; Wang, Ran; Zhang, Yunpeng; Zhang, Qian; Lu, Chengbiao

    2014-12-10

    The neurons of medial septal diagonal band of broca (MSDB) project to hippocampus and play an important role in MSDB-hippocampal synaptic transmission, plasticity and network oscillation. Nicotinic acetylcholine receptor (nAChR) subunits, α4β2 and α7 nAChRs, are expressed in MSDB neurons and permeable to calcium ions, which may modulate the function of MSDB neurons. The aims of this study are to determine the roles of selective nAChR activation on the calcium responses and membrane currents in MSDB neurons. Our results showed that nicotine increased calcium responses in the majority of MSDB neurons, pre-treatment of MSDB slices with a α4β2 nAChR antagonist, DhβE but not a α7 nAChR antagonist, MLA prevented nicotine-induced calcium responses. The whole cell patch clamp recordings showed that nicotine-induced inward current and acetylcholine (ACh) induced-firing activity can be largely reduced or prevented by DhβE in MSDB neurons. Surprisingly, post-treatment of α4β2 or α7 nAChR antagonists failed to block nicotine׳s role, they increased calcium responses instead. Application of calcium chelator EGTA reduced calcium responses in all neurons tested. These results suggest that there was a subtype specific modulation of nAChRs on calcium signaling and membrane currents in MSDB neurons and nAChR antagonists were also able to induce calcium responses involving a distinct mechanism.

  2. Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin

    Energy Technology Data Exchange (ETDEWEB)

    Byres, Emma; Paton, Adrienne W.; Paton, James C.; Löfling, Jonas C.; Smith, David F.; Wilce, Matthew C.J.; Talbot, Ursula M.; Chong, Damien C.; Yu, Hai; Huang, Shengshu; Chen, Xi; Varki, Nissi M.; Varki, Ajit; Rossjohn, Jamie; Beddoe, Travis (Emory-MED); (UCD); (Adelaide); (Monash)

    2009-01-30

    AB{sub 5} toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB{sub 5} toxin secreted by Shiga toxigenic Escherichia coli (STEC), which causes serious gastrointestinal disease in humans. SubAB causes haemolytic uraemic syndrome-like pathology in mice through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin's receptor is generated by metabolic incorporation of an exogenous factor derived from food.

  3. The nicotinic alpha7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-beta1-42

    DEFF Research Database (Denmark)

    Soderman, A.; Spang-Thomsen, Mogens; Hansen, H.

    2008-01-01

    7 nAChR have not been examined. The aim of this study has been to evaluate the efficacy of alpha7 nAChR modulators in transgene mice that overexpress human amyloid precursor protein and accumulate Abeta1-40 and Abeta1-42. In accordance with observations in human Alzheimer tissues, we show here...

  4. Kinase Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer

    Science.gov (United States)

    2017-02-01

    AWARD NUMBER: W81XWH-16-1-0009 TITLE: PRINCIPAL INVESTIGATOR: John Cleveland CONTRACTING ORGANIZATION: H. Lee Moffitt Cancer Center...so designated by other documentation. Kinase-Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer REPORT DOCUMENTATION PAGE Form...TITLE AND SUBTITLE 5a. CONTRACT NUMBER Kinase-Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer 5b. GRANT NUMBER W81XWH-16-1-0009 5c

  5. Kinase-Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer

    Science.gov (United States)

    2017-02-01

    AWARD NUMBER: W81XWH-16-1-0008 TITLE: Kinase-Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer PRINCIPAL INVESTIGATOR...Jan 2017 4. TITLE AND SUBTITLE Kinase-Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH...Investigator [PI], Scripps) and John Cleveland (Collaborating/Partnering PI, Moffitt Cancer Center) seek to validate 40S ribosome assembly as a therapeutic

  6. Possible neuroimmunomodulation therapy in T-cell-mediated oral diseases

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Sato

    2015-01-01

    Full Text Available Introduction: Recurrent aphthous stomatitis and oral lichen planus are local chronic inflammatory diseases which are implicated in T cell-mediated immunity. According to the systematic review, there is insufficient evidence to support any specific treatment for T-cell mediated oral diseases. The hypothesis: In this paper, we propose a hypothesis that recurrent aphthous stomatitis and oral lichen planus can be treated with selective α7 subunit of nicotinic acetylcholine receptor (α7 -nAChR agonists. Our hypothesis is supported by the following two facts. First, the pathophysiological conditions, T h 1/T h 17 cell activation and autonomic nervous system dysfunction, are observed in T-cell mediated oral diseases as well as in T-cell mediated systemic diseases such as rheumatoid arthritis. Second, the cholinergic anti-inflammatory pathway is inhibited in systemic T-cell mediated chronic inflammatory diseases. On the other hand, treatment with α7 -nAChR agonists which activate the cholinergic anti-inflammatory pathway suppresses neuroinflammation via inhibition of T h 1/T h 17 responses in animal model of systemic T-cell mediated chronic inflammatory diseases. We thus expect that selective α7 -nAChR agonists will be effective for the treatment of T-cell mediated oral diseases. Evaluation of the hypothesis: To test our hypothesis, we need to develop in vivo mouse model of T-cell mediated oral diseases. To evaluate the therapeutic effect of a selective α7 -nAChR agonist, we choose ABT-107 because of its safety and tolerability. We believe that the selective α7 -nAChR agonist, especially ABT-107, may be a therapeutic drug to treat T-cell mediated oral diseases.

  7. Nicotinic acetylcholine receptors (nAChRs) at zebrafish red and white muscle show different properties during development.

    Science.gov (United States)

    Ahmed, Kazi T; Ali, Declan W

    2016-08-01

    Nicotinic acetylcholine receptors (nAChRs) are highly expressed at the vertebrate neuromuscular junction (NMJ) where they are required for muscle activation. Understanding the factors that underlie NMJ development is critical for a full understanding of muscle function. In this study we performed whole cell and outside-out patch clamp recordings, and single-cell RT-qPCR from zebrafish red and white muscle to examine the properties of nAChRs during the first 5 days of development. In red fibers miniature endplate currents (mEPCs) exhibit single exponential time courses at 1.5 days postfertilization (dpf) and double exponential time courses from 2 dpf onwards. In white fibers, mEPCs decay relatively slowly, with a single exponential component at 1.5 dpf. By 2 and 3 dpf, mEPC kinetics speed up, and decay with a double exponential component, and by 4 dpf the exponential decay reverts back to a single component. Single channel recordings confirm the presence of two main conductance classes of nAChRs (∼45 pS and ∼65 pS) in red fibers with multiple time courses. Two main conductance classes are also present in white fibers (∼55 pS and ∼73 pS), but they exhibit shorter mean open times by 5 dpf compared with red muscle. RT-qPCR of mRNA for nicotinic receptor subunits supports a switch from γ to ε subunits in white fibers but not in red. Our findings provide a developmental profile of mEPC properties from red and white fibers in embryonic and larval zebrafish, and reveal previously unknown differences between the NMJs of these muscle fibers.© 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 916-936, 2016.

  8. Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs.

    Science.gov (United States)

    Wang, Yuexiang; Lee, Jang-Won; Oh, Gyeon; Grady, Sharon R; McIntosh, J Michael; Brunzell, Darlene H; Cannon, Jason R; Drenan, Ryan M

    2014-04-01

    α6β2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9'S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9'S NAc slices compared to control, non-transgenic (non-Tg) slices. Extracellular DA levels in both non-Tg and α6L9'S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9'S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9'S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid compared to non-Tg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9'S compared to non-Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9'S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels. © 2013 International Society for Neurochemistry.

  9. Cognitive improvement by activation of alpha7 nicotinic acetylcholine receptors: from animal models to human pathophysiology

    DEFF Research Database (Denmark)

    Thomsen, Morten S; Hansen, Henrik H; Timmerman, Daniel B

    2010-01-01

    AChR agonists improves learning, memory, and attentional function in variety of animal models, and pro-cognitive effects of alpha(7) nAChR agonists have recently been demonstrated in patients with schizophrenia or Alzheimer's disease. The alpha(7) nAChR desensitizes rapidly in vitro, and this has been a major...... concern in the development of alpha(7) nAChR agonists as putative drugs. Our review of the existing literature shows that development of tolerance to the behavioral effects of alpha(7) nAChR agonists does not occur in animal models or humans. However, the long-term memory-enhancing effects seen in animal...... models are not mimicked in healthy humans and schizophrenic patients, where attentional improvement predominates. This discrepancy may result from inherent differences in testing methods or from species differences in the level of expression of alpha(7) nAChRs in limbic brain regions, and may hamper...

  10. Serum inflammatory mediators as markers of human Lyme disease activity.

    Directory of Open Access Journals (Sweden)

    Mark J Soloski

    Full Text Available Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005 in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG, CXCL10 (IP-10 and CCL19 (MIP3B were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively. There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01 and the decrease correlates with chemokine levels (p = 0.0375. The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  11. Serum inflammatory mediators as markers of human Lyme disease activity.

    Science.gov (United States)

    Soloski, Mark J; Crowder, Lauren A; Lahey, Lauren J; Wagner, Catriona A; Robinson, William H; Aucott, John N

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005) in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG), CXCL10 (IP-10) and CCL19 (MIP3B) were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively). There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  12. Serum Inflammatory Mediators as Markers of Human Lyme Disease Activity

    Science.gov (United States)

    Soloski, Mark J.; Crowder, Lauren A.; Lahey, Lauren J.; Wagner, Catriona A.

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (pLyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations. PMID:24740099

  13. Cutting edge: Human regulatory T cells require IL-35 to mediate suppression and infectious tolerance.

    Science.gov (United States)

    Chaturvedi, Vandana; Collison, Lauren W; Guy, Clifford S; Workman, Creg J; Vignali, Dario A A

    2011-06-15

    Human regulatory T cells (T(reg)) are essential for the maintenance of immune tolerance. However, the mechanisms they use to mediate suppression remain controversial. Although IL-35 has been shown to play an important role in T(reg)-mediated suppression in mice, recent studies have questioned its relevance in human T(reg). In this study, we show that human T(reg) express and require IL-35 for maximal suppressive capacity. Substantial upregulation of EBI3 and IL12A, but not IL10 and TGFB, was observed in activated human T(reg) compared with conventional T cells (T(conv)). Contact-independent T(reg)-mediated suppression was IL-35 dependent and did not require IL-10 or TGF-β. Lastly, human T(reg)-mediated suppression led to the conversion of the suppressed T(conv) into iTr35 cells, an IL-35-induced T(reg) population, in an IL-35-dependent manner. Thus, IL-35 contributes to human T(reg)-mediated suppression, and its conversion of suppressed target T(conv) into IL-35-induced T(reg) may contribute to infectious tolerance.

  14. Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes.

    Science.gov (United States)

    Holubcová, Zuzana; Blayney, Martyn; Elder, Kay; Schuh, Melina

    2015-06-05

    Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs.

  15. Identification of novel α4β2-nicotinic acetylcholine receptor (nAChR) agonists based on an isoxazole ether scaffold that demonstrate antidepressant-like activity.

    Science.gov (United States)

    Yu, Li-Fang; Tückmantel, Werner; Eaton, J Brek; Caldarone, Barbara; Fedolak, Allison; Hanania, Taleen; Brunner, Dani; Lukas, Ronald J; Kozikowski, Alan P

    2012-01-26

    There is considerable evidence to support the hypothesis that the blockade of nAChR is responsible for the antidepressant action of nicotinic ligands. The nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine, has been shown to be an effective add-on in patients that do not respond to selective serotonin reuptake inhibitors. This suggests that nAChR ligands may address an unmet clinical need by providing relief from depressive symptoms in refractory patients. In this study, a new series of nAChR ligands based on an isoxazole-ether scaffold have been designed and synthesized for binding and functional assays. Preliminary structure-activity relationship (SAR) efforts identified a lead compound 43, which possesses potent antidepressant-like activity (1 mg/kg, IP; 5 mg/kg, PO) in the classical mouse forced swim test. Early stage absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) studies also suggested favorable drug-like properties, and broad screening toward other common neurotransmitter receptors indicated that compound 43 is highly selective for nAChRs over the other 45 neurotransmitter receptors and transporters tested.

  16. Crystal structure of Lymnaea stagnalis AChBP complexed with the potent nAChR antagonist DHβE suggests a unique mode of antagonism.

    Directory of Open Access Journals (Sweden)

    Azadeh Shahsavar

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed, activated (open, and desensitized (closed states. The acetylcholine binding protein (AChBP is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls in complex with dihydro-β-erythroidine (DHβE, which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.

  17. Dopamine in the medial amygdala network mediates human bonding

    Science.gov (United States)

    Touroutoglou, Alexandra; Rudy, Tali; Salcedo, Stephanie; Feldman, Ruth; Hooker, Jacob M.; Dickerson, Bradford C.; Catana, Ciprian; Barrett, Lisa Feldman

    2017-01-01

    Research in humans and nonhuman animals indicates that social affiliation, and particularly maternal bonding, depends on reward circuitry. Although numerous mechanistic studies in rodents demonstrated that maternal bonding depends on striatal dopamine transmission, the neurochemistry supporting maternal behavior in humans has not been described so far. In this study, we tested the role of central dopamine in human bonding. We applied a combined functional MRI-PET scanner to simultaneously probe mothers’ dopamine responses to their infants and the connectivity between the nucleus accumbens (NAcc), the amygdala, and the medial prefrontal cortex (mPFC), which form an intrinsic network (referred to as the “medial amygdala network”) that supports social functioning. We also measured the mothers’ behavioral synchrony with their infants and plasma oxytocin. The results of this study suggest that synchronous maternal behavior is associated with increased dopamine responses to the mother’s infant and stronger intrinsic connectivity within the medial amygdala network. Moreover, stronger network connectivity is associated with increased dopamine responses within the network and decreased plasma oxytocin. Together, these data indicate that dopamine is involved in human bonding. Compared with other mammals, humans have an unusually complex social life. The complexity of human bonding cannot be fully captured in nonhuman animal models, particularly in pathological bonding, such as that in autistic spectrum disorder or postpartum depression. Thus, investigations of the neurochemistry of social bonding in humans, for which this study provides initial evidence, are warranted. PMID:28193868

  18. BST-2 mediated restriction of simian-human immunodeficiency virus.

    Science.gov (United States)

    Ruiz, Autumn; Lau, David; Mitchell, Richard S; Hill, M Sarah; Schmitt, Kimberly; Guatelli, John C; Stephens, Edward B

    2010-10-25

    Pathogenic simian-human immunodeficiency viruses (SHIV) contain HIV-1 Vpu and SIV Nef, both shown to counteract BST-2 (HM1.24; CD317; tetherin) inhibition of virus release in a species-specific manner. We show that human and pig-tailed BST-2 (ptBST-2) restrict SHIV. We found that sequential "humanization" of the transmembrane domain (TMD) of the pig-tailed BST-2 (ptBST-2) protein resulted in a fluctuation in sensitivity to HIV-1 Vpu. Our results also show that the length of the TMD in human and ptBST-2 proteins is important for BST-2 restriction and susceptibility to Vpu. Taken together, our results emphasize the importance of tertiary structure in BST-2 antagonism and suggests that the HIV-1 Vpu transmembrane domain may have additional functions in vivo unrelated to BST-2 antagonism.

  19. Comparison of epigenetic mediator expression and function in mouse and human embryonic blastomeres

    OpenAIRE

    Chavez, Shawn L.; McElroy, Sohyun L.; Bossert, Nancy L.; De Jonge, Christopher J.; Rodriguez, Maria Vera; Denise E Leong; Behr, Barry; Westphal, Lynn M.; Reijo Pera, Renee A.

    2014-01-01

    A map of human embryo development that combines imaging, molecular, genetic and epigenetic data for comparisons to other species and across pathologies would be greatly beneficial for basic science and clinical applications. Here, we compared mRNA and protein expression of key mediators of DNA methylation and histone modifications between mouse and human embryos, embryos from fertile/infertile couples, and following growth factor supplementation. We observed that individual mouse and human em...

  20. Self-organizing human cardiac microchambers mediated by geometric confinement

    Science.gov (United States)

    Ma, Zhen; Wang, Jason; Loskill, Peter; Huebsch, Nathaniel; Koo, Sangmo; Svedlund, Felicia L.; Marks, Natalie C.; Hua, Ethan W.; Grigoropoulos, Costas P.; Conklin, Bruce R.; Healy, Kevin E.

    2015-07-01

    Tissue morphogenesis and organ formation are the consequences of biochemical and biophysical cues that lead to cellular spatial patterning in development. To model such events in vitro, we use PEG-patterned substrates to geometrically confine human pluripotent stem cell colonies and spatially present mechanical stress. Modulation of the WNT/β-catenin pathway promotes spatial patterning via geometric confinement of the cell condensation process during epithelial-mesenchymal transition, forcing cells at the perimeter to express an OCT4+ annulus, which is coincident with a region of higher cell density and E-cadherin expression. The biochemical and biophysical cues synergistically induce self-organizing lineage specification and creation of a beating human cardiac microchamber confined by the pattern geometry. These highly defined human cardiac microchambers can be used to study aspects of embryonic spatial patterning, early cardiac development and drug-induced developmental toxicity.

  1. Adaptations to climate-mediated selective pressures in humans.

    Directory of Open Access Journals (Sweden)

    Angela M Hancock

    2011-04-01

    Full Text Available Humans inhabit a remarkably diverse range of environments, and adaptation through natural selection has likely played a central role in the capacity to survive and thrive in extreme climates. Unlike numerous studies that used only population genetic data to search for evidence of selection, here we scan the human genome for selection signals by identifying the SNPs with the strongest correlations between allele frequencies and climate across 61 worldwide populations. We find a striking enrichment of genic and nonsynonymous SNPs relative to non-genic SNPs among those that are strongly correlated with these climate variables. Among the most extreme signals, several overlap with those from GWAS, including SNPs associated with pigmentation and autoimmune diseases. Further, we find an enrichment of strong signals in gene sets related to UV radiation, infection and immunity, and cancer. Our results imply that adaptations to climate shaped the spatial distribution of variation in humans.

  2. Targeting α4β2 nAChRs in CNS disorders: Perspectives on positive allosteric modulation as a therapeutic approach

    DEFF Research Database (Denmark)

    Grupe, Morten; Grunnet, Morten; Bastlund, Jesper F.;

    2015-01-01

    The nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels broadly involved in regulating neurotransmission in the central nervous system (CNS) by conducting cation currents through the membrane of neurons. Many different nAChR subtypes exist with each their functional...... characteristics, expression pattern and pharmacological profile. The focus of the present MiniReview is on the heteromeric α4β2 nAChR, as activity at this subtype contributes to cognitive functioning through interactions with multiple neurotransmitter systems and is implicated in various CNS disorders...... be used as a treatment approach in various CNS disorders. As subtype-selective agonists and other cholinergic ligands have only shown limited therapeutic success, the focus of recent drug development endeavours has largely shifted to positive allosteric modulators (PAMs). By potentiating the action...

  3. Fcgamma receptor-mediated suppression of human immunodeficiency virus type 1 replication in primary human macrophages.

    Science.gov (United States)

    Perez-Bercoff, Danielle; David, Annie; Sudry, Hugues; Barré-Sinoussi, Françoise; Pancino, Gianfranco

    2003-04-01

    Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is modulated by various stimuli. In this study we demonstrate that stimulation of primary monocytes and monocyte-derived macrophages (MDM) through the receptors for the Fc portion of immunoglobulin G (IgG) (FcgammaR) inhibits HIV type 1 (HIV-1) replication. Viral p24 production was decreased by 1.5 to 3 log units in MDM infected with both R5 and X4 HIV-1 strains upon stimulation by immobilized IgG but not upon stimulation by soluble IgG or by F(ab')(2) IgG fragments. Although MDM activation by immobilized IgG induced high levels of macrophage-derived chemokine secretion as well as a sustained down-regulation of CD4 and a transient decrease in CCR5 expression, these factors did not appear to play a major role in the suppression of HIV-1 replication. Single-cycle infection of FcgammaR-stimulated MDM with HIV-1 virions pseudotyped with either HIV-1 R5 or vesicular stomatitis virus G envelopes was inhibited, suggesting a postentry restriction of viral replication. PCR analyses of HIV-1 DNA intermediate replication forms suggested that reverse transcription is not affected by stimulation with immobilized human IgG, at least during the first replication cycle. The accumulation of PCR products corresponding to nuclear unintegrated two-long-terminal-repeat circles and the relative decrease of integrated HIV-1 DNA signals suggest an inhibition of proviral integration. Our data, showing that FcgammaR-mediated activation of MDM is a potent mechanism of HIV-1 suppression, raise the possibility that FcgammaR cross-linking by immune complexes may contribute to the control of viral replication in macrophages.

  4. HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate whether the non-classi-cal HLA-G classⅠmolecule protects the porcine endothelial cells (PECs) from the lysis mediated by human immune cells in pig to human discordant xenotransplantation, we have cloned HLA-G cDNA from a human placenta by RT-PCR. Mammalian expression vector, pEFG-neo, was constructed by insertion of HLA-G cDNA in pEF-neo. We obtained efficiently expressed PECs by stable transfection. Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis. The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells. It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes. The reduction of lysis ranged between 59.1% and 88.9%. These findings suggest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overcoming the immune rejections in xenotransplantion, including delayed xenograft rejection and cell-mediated rejection.

  5. Human shields mediate sexual conflict in a top predator

    Science.gov (United States)

    Pelletier, F.; Kindberg, J.; Brunberg, S.; Swenson, J. E.; Zedrosser, A.

    2016-01-01

    Selecting the right habitat in a risky landscape is crucial for an individual's survival and reproduction. In predator–prey systems, prey often can anticipate the habitat use of their main predator and may use protective associates (i.e. typically an apex predator) as shields against predation. Although never tested, such mechanisms should also evolve in systems in which sexual conflict affects offspring survival. Here, we assessed the relationship between offspring survival and habitat selection, as well as the use of protective associates, in a system in which sexually selected infanticide (SSI), rather than interspecific predation, affects offspring survival. We used the Scandinavian brown bear (Ursus arctos) population with SSI in a human-dominated landscape as our model system. Bears, especially adult males, generally avoid humans in our study system. We used resource selection functions to contrast habitat selection of GPS-collared mothers that were successful (i.e. surviving litters, n = 19) and unsuccessful (i.e. complete litter loss, n = 11) in keeping their young during the mating season (2005–2012). Habitat selection was indeed a predictor of litter survival. Successful mothers were more likely to use humans as protective associates, whereas unsuccessful mothers avoided humans. Our results suggest that principles of predator–prey and fear ecology theory (e.g. non-consumptive and cascading effects) can also be applied to the context of sexual conflict. PMID:27335423

  6. Nanoparticle (MPG)-mediated delivery of small RNAs into human ...

    African Journals Online (AJOL)

    Aghomotsegin

    stranded siRNA into human multipotent stem (hMADS) cells. Confluent and .... sequence, which then adopted a β-structure required for insertion of the peptide into the ... confocal laser microscopy they observed a punctual intracellular pattern ...

  7. UVA Light-mediated Ascorbate Oxidation in Human Lenses.

    Science.gov (United States)

    Rakete, Stefan; Nagaraj, Ram H

    2017-01-13

    Whether ascorbate oxidation is promoted by UVA light in human lenses and whether this process is influenced by age and GSH levels are not known. In this study, we used paired lenses from human donors. One lens of each pair was exposed to UVA light, whereas the other lens was kept in the dark for the same period of time as the control. Using LC-MS/MS analyses, we found that older lenses (41-73 years) were more susceptible to UVA-induced ascorbate oxidation than younger lenses (18-40 years). Approximately 36% of the ascorbate (relative to control) was oxidized in older lenses compared to ~16% in younger lenses. Furthermore, lenses with higher levels of GSH were less susceptible to UVA-induced ascorbate oxidation compared to those with lower levels, and this effect was not dependent on age. The oxidation of ascorbate led to elevated levels of reactive α-dicarbonyl compounds. In summary, our study showed that UVA light exposure leads to ascorbate oxidation in human lenses and that such oxidation is more pronounced in aged lenses and is inversely related to GSH levels. Our findings suggest that UVA light exposure could lead to protein aggregation through ascorbate oxidation in human lenses.

  8. Improvement of Radiation-Mediated Immunosuppression of Human NSCLC Tumour Xenografts in a Nude Rat Model

    Directory of Open Access Journals (Sweden)

    Sergey V. Tokalov

    2010-01-01

    Full Text Available Human tumour xenografts in a nude rat model have consistently been used as an essential part of preclinical studies for anticancer drugs activity in human. Commonly, these animals receive whole body irradiation to assure immunosuppression. But whole body dose delivery might be inhomogeneous and the resulting incomplete bone marrow depletion may modify tumour behaviour. To improve irradiation-mediated immunosuppression of human non-small cell lung cancer (NSCLC xenografts in a nude rat model irradiation (2 + 2 Gy from opposite sides of animals has been performed using a conventional X-ray tube. The described modification of whole body irradiation improves growth properties of human NSCLC xenografts in a nude rat model. The design of the whole body irradiation mediated immunosuppression described here for NSCLC xenografts may be useful for research applications involving other types of human tumours.

  9. Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida

    Directory of Open Access Journals (Sweden)

    Talwar Pankaj

    2010-01-01

    Full Text Available Abstract Background Human egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP, responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction. Methods Heat-solubilized human ZP (SIZP was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP. Results SIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl- channels play an important role in SIZP mediated induction of acrosome reaction. Conclusions Results described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.

  10. The Signature of Selection Mediated by Expression on Human Genes

    OpenAIRE

    Urrutia, Araxi O.; Hurst, Laurence D

    2003-01-01

    As the efficacy of natural selection is expected to be a function of population size, in humans it is usually presumed that selection is a weak force and hence that gene characteristics are mostly determined by stochastic forces. In contrast, in species with large population sizes, selection is expected to be a much more effective force. Evidence for this has come from examining how genic parameters vary with expression level, which appears to determine many of a gene's features, such as codo...

  11. Menin: a tumor suppressor that mediates postsynaptic receptor expression and synaptogenesis between central neurons of Lymnaea stagnalis.

    Directory of Open Access Journals (Sweden)

    Nichole Flynn

    Full Text Available Neurotrophic factors (NTFs support neuronal survival, differentiation, and even synaptic plasticity both during development and throughout the life of an organism. However, their precise roles in central synapse formation remain unknown. Previously, we demonstrated that excitatory synapse formation in Lymnaea stagnalis requires a source of extrinsic NTFs and receptor tyrosine kinase (RTK activation. Here we show that NTFs such as Lymnaea epidermal growth factor (L-EGF act through RTKs to trigger a specific subset of intracellular signalling events in the postsynaptic neuron, which lead to the activation of the tumor suppressor menin, encoded by Lymnaea MEN1 (L-MEN1 and the expression of excitatory nicotinic acetylcholine receptors (nAChRs. We provide direct evidence that the activation of the MAPK/ERK cascade is required for the expression of nAChRs, and subsequent synapse formation between pairs of neurons in vitro. Furthermore, we show that L-menin activation is sufficient for the expression of postsynaptic excitatory nAChRs and subsequent synapse formation in media devoid of NTFs. By extending our findings in situ, we reveal the necessity of EGFRs in mediating synapse formation between a single transplanted neuron and its intact presynaptic partner. Moreover, deficits in excitatory synapse formation following EGFR knock-down can be rescued by injecting synthetic L-MEN1 mRNA in the intact central nervous system. Taken together, this study provides the first direct evidence that NTFs functioning via RTKs activate the MEN1 gene, which appears sufficient to regulate synapse formation between central neurons. Our study also offers a novel developmental role for menin beyond tumour suppression in adult humans.

  12. Menin: A Tumor Suppressor That Mediates Postsynaptic Receptor Expression and Synaptogenesis between Central Neurons of Lymnaea stagnalis

    Science.gov (United States)

    Flynn, Nichole; Getz, Angela; Visser, Frank; Janes, Tara A.; Syed, Naweed I.

    2014-01-01

    Neurotrophic factors (NTFs) support neuronal survival, differentiation, and even synaptic plasticity both during development and throughout the life of an organism. However, their precise roles in central synapse formation remain unknown. Previously, we demonstrated that excitatory synapse formation in Lymnaea stagnalis requires a source of extrinsic NTFs and receptor tyrosine kinase (RTK) activation. Here we show that NTFs such as Lymnaea epidermal growth factor (L-EGF) act through RTKs to trigger a specific subset of intracellular signalling events in the postsynaptic neuron, which lead to the activation of the tumor suppressor menin, encoded by Lymnaea MEN1 (L-MEN1) and the expression of excitatory nicotinic acetylcholine receptors (nAChRs). We provide direct evidence that the activation of the MAPK/ERK cascade is required for the expression of nAChRs, and subsequent synapse formation between pairs of neurons in vitro. Furthermore, we show that L-menin activation is sufficient for the expression of postsynaptic excitatory nAChRs and subsequent synapse formation in media devoid of NTFs. By extending our findings in situ, we reveal the necessity of EGFRs in mediating synapse formation between a single transplanted neuron and its intact presynaptic partner. Moreover, deficits in excitatory synapse formation following EGFR knock-down can be rescued by injecting synthetic L-MEN1 mRNA in the intact central nervous system. Taken together, this study provides the first direct evidence that NTFs functioning via RTKs activate the MEN1 gene, which appears sufficient to regulate synapse formation between central neurons. Our study also offers a novel developmental role for menin beyond tumour suppression in adult humans. PMID:25347295

  13. Converting enzyme inhibitor temocaprilat prevents high glucose-mediated suppression of human aortic endothelial cell proliferation.

    Science.gov (United States)

    Yasunari, Kenichi; Maeda, Kensaku; Watanabe, Takanori; Nakamura, Munehiro; Asada, Akira; Yoshikawa, Junichi

    2003-12-01

    We examined the involvement of the oxidative stress in high glucose-induced suppression of human aortic endothelial cell proliferation. Chronic glucose treatment for 72 h concentration-dependently (5.6-22.2 mol/l) inhibited human coronary endothelial cell proliferation. Temocaprilat, an angiotensin-converting enzyme inhibitor, at 10 nmol/l to 1 micromol/l inhibited high glucose (22.2 mmol/l)-mediated suppression of human aortic endothelial cell proliferation. Temocaprilat at 1 micromol/l inhibited high glucose-induced membrane-bound protein kinase C activity in human aortic endothelial cells. The protein kinase C inhibitors calphostin C 100 nmol/l or chelerythrine 1 micromol/l inhibited high glucose-mediated suppression of human aortic endothelial cell proliferation. Chronic high glucose treatment for 72 h increased intracellular oxidative stress, directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by temocaprilat 10 nmol/l to 1 micromol/l. Bradykinin B2 receptor antagonist icatibant 100 nmol/l significantly reduced the action of temocaprilat; whereas bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin 100 nmol/l had no effect. These findings suggest that high glucose inhibits human aortic endothelial cell proliferation and that the angiotensin-converting enzyme inhibitor temocaprilat inhibits high glucose-mediated suppression of human aortic endothelial cell proliferation, possibly through suppression of protein kinase C, bradykinin B2 receptors and oxidative stress.

  14. Mercury induces inflammatory mediator release from human mast cells

    Directory of Open Access Journals (Sweden)

    Peterson Erika

    2010-03-01

    Full Text Available Abstract Background Mercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl2 on human mast cell activation. Methods Human leukemic cultured LAD2 mast cells and normal human umbilical cord blood-derived cultured mast cells (hCBMCs were stimulated by HgCl2 (0.1-10 μM for either 10 min for beta-hexosaminidase release or 24 hr for measuring vascular endothelial growth factor (VEGF and IL-6 release by ELISA. Results HgCl2 induced a 2-fold increase in β-hexosaminidase release, and also significant VEGF release at 0.1 and 1 μM (311 ± 32 pg/106 cells and 443 ± 143 pg/106 cells, respectively from LAD2 mast cells compared to control cells (227 ± 17 pg/106 cells, n = 5, p 2 (0.1 μM to the proinflammatory neuropeptide substance P (SP, 0.1 μM had synergestic action in inducing VEGF from LAD2 mast cells. HgCl2 also stimulated significant VEGF release (360 ± 100 pg/106 cells at 1 μM, n = 5, p 6 cells, and IL-6 release (466 ± 57 pg/106 cells at 0.1 μM compared to untreated cells (13 ± 25 pg/106 cells, n = 5, p 2 (0.1 μM to SP (5 μM further increased IL-6 release. Conclusions HgCl2 stimulates VEGF and IL-6 release from human mast cells. This phenomenon could disrupt the blood-brain-barrier and permit brain inflammation. As a result, the findings of the present study provide a biological mechanism for how low levels of mercury may contribute to ASD pathogenesis.

  15. Human cell mediated immunity to porins from Salmonella typhi.

    Science.gov (United States)

    Blanco, F; Isibasi, A; Raúl González, C; Ortiz, V; Paniagua, J; Arreguín, C; Kumate, J

    1993-01-01

    The current studies were undertaken to assess the role of the porins and outer membrane proteins (OMP) in the human immune response to Salmonella typhi 9, 12 Vi:d. Experiments were performed to determinate the lymphocyte activation response to porins in individuals who had been vaccinated against typhoid fever. 10 healthy volunteers were studied before and 10 days after oral or subcutaneous immunisation. Five patients with typhoid fever were also studied. Lymphocyte activation was measured by the 3H thymidine incorporation assay. Individuals with typhoid fever as well as those immunised with oral vaccine responded well to porins and outer membrane proteins, as opposed to those immunised with the subcutaneous vaccine. These results suggest that the porins and OMP play a role in the cellular immune response against Salmonella typhi.

  16. Mechanism of human antibody-mediated neutralization of Marburg virus.

    Science.gov (United States)

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  17. CRISPR-mediated genome editing and human diseases

    Directory of Open Access Journals (Sweden)

    Liquan Cai

    2016-12-01

    Full Text Available CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats technology has emerged as a powerful technology for genome editing and is now widely used in basic biomedical research to explore gene function. More recently, this technology has been increasingly applied to the study or treatment of human diseases, including Barth syndrome effects on the heart, Duchenne muscular dystrophy, hemophilia, β-Thalassemia, and cystic fibrosis. CRISPR/Cas9 (CRISPR-associated protein 9 genome editing has been used to correct disease-causing DNA mutations ranging from a single base pair to large deletions in model systems ranging from cells in vitro to animals in vivo. In addition to genetic diseases, CRISPR/Cas9 gene editing has also been applied in immunology-focused applications such as the targeting of C-C chemokine receptor type 5, the programmed death 1 gene, or the creation of chimeric antigen receptors in T cells for purposes such as the treatment of the acquired immune deficiency syndrome (AIDS or promoting anti-tumor immunotherapy. Furthermore, this technology has been applied to the genetic manipulation of domesticated animals with the goal of producing biologic medical materials, including molecules, cells or organs, on a large scale. Finally, CRISPR/Cas9 has been teamed with induced pluripotent stem (iPS cells to perform multiple tissue engineering tasks including the creation of disease models or the preparation of donor-specific tissues for transplantation. This review will explore the ways in which the use of CRISPR/Cas9 is opening new doors to the treatment of human diseases.

  18. Lovastatin-Mediated Changes in Human Tendon Cells.

    Science.gov (United States)

    Kuzma-Kuzniarska, Maria; Cornell, Hannah R; Moneke, Michael C; Carr, Andrew J; Hulley, Philippa A

    2015-10-01

    Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol-lowering and anti-atherosclerotic properties. Although some statin patients may experience muscle-related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first-pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects; however, recently some statin-related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon-specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short-term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability; however, cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP-2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose-dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function.

  19. Nuclear factor-κB mediates placental growth factor induced pro-labour mediators in human placenta.

    Science.gov (United States)

    Lappas, Martha

    2012-07-01

    Prostaglandins, pro-inflammatory cytokines, extracellular matrix remodelling enzymes and nuclear factor-kappa B (NF-κB) are involved in the mechanisms of term and preterm parturition. Recent studies have reported an increase in angiogenesis-related genes during term and preterm labour, including placental growth factor (PLGF). In non-gestational tissues, PLGF induces inflammation via NF-κB. The aim of this study was to determine the effect of PLGF on the gene expression and release of pro-labour mediators in human placenta. Samples were obtained from normal pregnancies at the time of Caesarean section. Human placenta was incubated in the absence (basal control) or presence of a 10 ng/ml PLGF for 24 h. Inflammatory gene expression was analysed by quantitative RT-PCR, concentration of pro-inflammatory cytokines and prostaglandins was quantified by ELISA, and secretory matrix metalloproteinases (MMPs) activity by zymography. NF-κB DNA-binding activity and IκB-α (inhibitor of NF-κB) protein degradation were analysed by ELISA and Western blotting, respectively. PLGF significantly increased interleukin (IL)-6 and IL-8 gene expression and secretion, cyclooxygenase-2 expression and resultant prostaglandin (PG) E(2) and PGF(2α) release, and MMP-9 gene expression and enzyme production. PLGF induced the degradation of IκB-α whilst increasing NF-κB p65 DNA-binding activity. The PLGF-induced pro-labour responses were abrogated by co-treatment with the NF-κB inhibitor BAY 11-7082. In summary, the pro-inflammatory and pro-labour effects of PLGF in human placenta are mediated by NF-κB.

  20. Palindrome-mediated Translocations in Humans: A New Mechanistic Model for Gross Chromosomal Rearrangements

    Directory of Open Access Journals (Sweden)

    Hidehito Inagaki

    2016-07-01

    Full Text Available Palindromic DNA sequences, which can form secondary structures, are widely distributed in the human genome. Although the nature of the secondary structure—single-stranded hairpin or double-stranded cruciform—has been extensively investigated in vitro, the existence of such unusual non-B DNA in vivo remains controversial. Here, we review palindrome-mediated gross chromosomal rearrangements possibly induced by non-B DNA in humans. Recent advances in next-generation sequencing have not yet overcome the difficulty of palindromic sequence analysis. However, a dozen palindromic AT-rich repeat (PATRR sequences have been identified at the breakpoints of recurrent or non-recurrent chromosomal translocations in humans. The breakages always occur at the center of the palindrome. Analyses of polymorphisms within the palindromes indicate that the symmetry and length of the palindrome affect the frequency of the de novo occurrence of these palindrome-mediated translocations, suggesting the involvement of non-B DNA. Indeed, experiments using a plasmid-based model system showed that the formation of non-B DNA is likely the key to palindrome-mediated genomic rearrangements. Some evidence implies a new mechanism that cruciform DNAs may come close together first in nucleus and illegitimately joined. Analysis of PATRR-mediated translocations in humans will provide further understanding of gross chromosomal rearrangements in many organisms.

  1. Increase human metapneumovirus mediated morbidity following pandemic influenza infection.

    Directory of Open Access Journals (Sweden)

    Liora Regev

    Full Text Available Human metapneumovirus (hMPV is a recently discovered respiratory pathogen, infecting mainly young children. The infected patients suffer from influenza like symptoms (ILS. In Israel the virus is mainly circulating in February to March. Here we report on an increased rate of hMPV infection in the winter season of 2009-10. The 2009-10 infection had several unique characteristics when compared to previous seasons; it started around January and a large number of infants were infected by the virus. Genetic analysis based on the viral L and F genes of hMPV showed that only subtypes A2 and B2 circulated in Israel. Additionally, we have identified a novel variant of hMPV within subgroup A2b, which subdivide it into A2b1 and A2b2. Finally, we showed that the hMPV infection was detected in the country soon after the infection with the pandemic influenza virus had declined, that infection with the pandemic influenza virus was dominant and that it interfered with the infection of other respiratory viruses. Thus, we suggest that the unusual increase in hMPV infection observed in 2009-10 was due to the appearance of the pandemic influenza virus in the winter season prior to 2009-10.

  2. Transposon-mediated BAC transgenesis in human ES cells.

    Science.gov (United States)

    Rostovskaya, Maria; Fu, Jun; Obst, Mandy; Baer, Isabell; Weidlich, Stefanie; Wang, Hailong; Smith, Andrew J H; Anastassiadis, Konstantinos; Stewart, A Francis

    2012-10-01

    Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs.

  3. GAGE12 mediates human gastric carcinoma growth and metastasis.

    Science.gov (United States)

    Lee, Eun Kyung; Song, Kyung-A; Chae, Ji-Hye; Kim, Kyoung-Mee; Kim, Seok-Hyung; Kang, Myung-Soo

    2015-05-15

    The spontaneous metastasis from human gastric carcinoma (GC) remains poorly reproduced in animal models. Here, we established an experimental mouse model in which GC progressively developed in the orthotopic stomach wall and metastasized to multiple organs; the tumors colonized in the ovary exhibited typical characteristics of Krukenberg tumor. The expression of mesenchymal markers was low in primary tumors and high in those in intravasating and extravasating veins. However, the expression of epithelial markers did not differ, indicating that the acquisition of mesenchymal markers without a concordant loss of typical epithelial markers was associated with metastasis. We identified 35 differentially expressed genes (DEGs) in GC cells metastasized to ovary, among which overexpression of GAGE12 family genes, the top-ranked DEGs, were validated. In addition, knockdown of the GAGE12 gene family affected transcription of many of the aforementioned 35 DEGs and inhibited trans-well migration, tumor sphere formation in vitro and tumor growth in vivo. In accordance, GAGE12 overexpression augmented migration, tumor sphere formation and sustained in vivo tumor growth. Taken together, the GAGE12 gene family promotes GC growth and metastasis by modulating the expression of GC metastasis-related genes.

  4. Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly.

    Directory of Open Access Journals (Sweden)

    Carrie Bernecky

    2011-03-01

    Full Text Available The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC, consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II enzyme. The structure of the human Mediator-pol II interface is not well-characterized, whereas attempts to structurally define the Mediator-pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator-pol II-TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator-pol II-TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft are exposed within the Mediator-pol II-TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator-CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator-pol II complexes lacking TFIIF reveal that TFIIF plays a key role in

  5. Crystal Structure of Lymnaea stagnalis AChBP Complexed with the Potent nAChR Antagonist DHßE Suggests a Unique Mode of Antagonism

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Kastrup, Jette S; Nielsen, Elsebet Ø.;

    2012-01-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states...

  6. Binding of long-chain α-neurotoxin would stabilize the resting state of nAChR: A comparative study with α-conotoxin

    Directory of Open Access Journals (Sweden)

    Naderi-Manesh Hossein

    2009-02-01

    Full Text Available Abstract Background The details of interaction in a complex between potent antagonists such as long chain α-neurotoxins and α-conotoxins with nicotinic acetylcholine receptor (nAChR, and conformational changes induced by these antagonists, are not yet clear. Modeling In order to uncover some of these critical structural features, we conducted a docking simulation and a molecular dynamics simulation (MD of a model of the ligand binding domain of nAChR in complex with a long-chain α-neurotoxin and an α-conotoxin. Results Our docking results confirm the claim that T.nAChR is in the basal or resting state, which favors binding to the alpha-neurotoxins. Moreover, more correct "hits" for the α/γ interface upon docking for conotoxin-nAChR confirm the preference of conotoxin GI for the α/γ interface. More importantly, upon binding of α-neurotoxin, ligand-bonded nAChR is less dynamic in certain domains than the apo form of the conotoxin-AChR complex. Some critical interactions in the binding site such as the salt bridge formed between K145/D200 in the neurotoxin-nAChR complex is further stabilized during the MD simulation, while it is obviously more labile in the apo form. Conclusion These observations could support the claim that alpha neurotoxins stabilize the nAChR resting state.

  7. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.

    Science.gov (United States)

    Lee, Sahmin; Lee, Hyun-Chae; Kwon, Yoo-Wook; Lee, Sang Eun; Cho, Youngjin; Kim, Joonoh; Lee, Soobeom; Kim, Ju-Young; Lee, Jaewon; Yang, Han-Mo; Mook-Jung, Inhee; Nam, Ky-Youb; Chung, Junho; Lazar, Mitchell A; Kim, Hyo-Soo

    2014-03-04

    Human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. Resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. Nevertheless, the receptor for human resistin has not been clarified. Here, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We found that human resistin directly binds to CAP1 in monocytes and upregulates cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines. Overexpression of CAP1 in monocytes enhanced the resistin-induced increased activity of the cAMP-dependent signaling. Moreover, CAP1-overexpressed monocytes aggravated adipose tissue inflammation in transgenic mice that express human resistin from their monocytes. In contrast, suppression of CAP1 expression abrogated the resistin-mediated inflammatory activity both in vitro and in vivo. Therefore, CAP1 is the bona fide receptor for resistin leading to inflammation in humans.

  8. Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells.

    Science.gov (United States)

    Liu, Jing; Lange, Miles D; Hong, Sang Yong; Xie, Wanqin; Xu, Kerui; Huang, Lin; Yu, Yangsheng; Ehrhardt, Götz R A; Zemlin, Michael; Burrows, Peter D; Su, Kaihong; Carter, Robert H; Zhang, Zhixin

    2013-06-01

    VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

  9. Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics

    Directory of Open Access Journals (Sweden)

    Zachary C. Poss

    2016-04-01

    Full Text Available Cortistatin A (CA is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116. We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0–24 hr, revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair.

  10. Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

    Science.gov (United States)

    Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

    2016-04-26

    The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

  11. Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens

    Science.gov (United States)

    Casson, Cierra N.; Yu, Janet; Reyes, Valeria M.; Taschuk, Frances O.; Yadav, Anjana; Copenhaver, Alan M.; Nguyen, Hieu T.; Collman, Ronald G.; Shin, Sunny

    2015-01-01

    Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages. PMID:25964352

  12. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Singaravelu, Ragunath [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Lyn, Rodney K. [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Srinivasan, Prashanth [National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Delcorde, Julie [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Steenbergen, Rineke H.; Tyrrell, D. Lorne [Department of Medical Microbiology and Immunology, University of Alberta (Canada); Li Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, Edmonton, Alberta T6G 2S2 (Canada); Pezacki, John P., E-mail: John.Pezacki@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada)

    2013-11-15

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

  13. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans.

    Science.gov (United States)

    Lammers, Laureen A; Achterbergh, Roos; de Vries, Emmely M; van Nierop, F Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R; Boelen, Anita; Romijn, Johannes A; Mathôt, Ron A A

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans.

  14. Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells.

    Science.gov (United States)

    Jin, Un-Ho; Song, Kwon-Ho; Motomura, Muneo; Suzuki, Ikukatsu; Gu, Yeun-Hwa; Kang, Yun-Jeong; Moon, Tae-Chul; Kim, Cheorl-Ho

    2008-03-01

    Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 microg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2 alpha and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells.

  15. Impaired cell mediated immunity in haemophilia in the absence of infection with human immunodeficiency virus.

    Science.gov (United States)

    Madhok, R; Gracie, A; Lowe, G D; Burnett, A; Froebel, K; Follett, E; Forbes, C D

    1986-10-18

    The cell mediated immune response was evaluated in vivo in 29 patients with clinically severe haemophilia by means of the dinitrochlorobenzene skin test. All patients had a response below the median normal value, and in 19 the response was on or below the lower limit of the normal range. There was no difference in skin response between patients positive and negative for the human immunodeficiency virus (HIV; formerly known as human T cell lymphotropic virus III or lymphadenopathy associated virus). In the whole group, and in seronegative patients (n = 17), there was an inverse relation between exposure to clotting factor and skin response. In seropositive patients (n = 12) no such association was apparent. This study shows that clotting factor concentrate impairs the cell mediated immune response to a new antigen in the absence of infection with HIV.

  16. Sustained Small Interfering RNA-Mediated Human Immunodeficiency Virus Type 1 Inhibition in Primary Macrophages

    OpenAIRE

    2003-01-01

    Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor...

  17. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

    Science.gov (United States)

    Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2015-03-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

  18. HABP2 is a novel regulator of hyaluronan-mediated human lung cancer progression

    OpenAIRE

    Tamara eMirzapoiazova; Nurbek eMambetsariev; Frances E Lennon; Bolot eMambetsariev; Joshua E. Berlind; Ravi eSalgia; Singleton, Patrick A.

    2015-01-01

    Background: Lung cancer is a devastating disease with limited treatment options. Many lung cancers have changes in their microenvironment including upregulation of the extracellular matrix glycosaminoglycan, hyaluronan (HA), which we have previously demonstrated can regulate the activity of the extracellular serine protease, Hyaluronan Binding Protein 2 (HABP2). This study examined the functional role of HABP2 on HA-mediated human lung cancer dynamics.Methods: Immunohistochemical analysis was...

  19. HABP2 is a Novel Regulator of Hyaluronan-Mediated Human Lung Cancer Progression

    OpenAIRE

    Mirzapoiazova, Tamara; Mambetsariev, Nurbek; Frances E Lennon; Mambetsariev, Bolot; Joshua E. Berlind; Salgia, Ravi; Singleton, Patrick A.

    2015-01-01

    Background Lung cancer is a devastating disease with limited treatment options. Many lung cancers have changes in their microenvironment including upregulation of the extracellular matrix glycosaminoglycan, hyaluronan (HA), which we have previously demonstrated can regulate the activity of the extracellular serine protease, hyaluronan binding protein 2 (HABP2). This study examined the functional role of HABP2 on HA-mediated human lung cancer dynamics. Methods Immunohistochemical an...

  20. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

    Institute of Scientific and Technical Information of China (English)

    Nasrin NFJATBAKHSH; Zhong-ping FENG

    2011-01-01

    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

  1. Capsular polysaccharide of Group B Streptococcus mediates biofilm formation in the presence of human plasma.

    Science.gov (United States)

    Xia, Fan Di; Mallet, Adeline; Caliot, Elise; Gao, Cherry; Trieu-Cuot, Patrick; Dramsi, Shaynoor

    2015-01-01

    Group B Streptococcus (GBS) is an asymptomatic colonizer of human mucosal surfaces that is responsible for sepsis and meningitis in neonates. Bacterial persistence and pathogenesis often involves biofilm formation. We previously showed that biofilm formation in medium supplemented with glucose is mediated by the PI-2a pilus. Here, biofilm formation was tested in cell culture medium supplemented with human plasma. GBS strains were able to form biofilms in these conditions unlike Group A Streptococcus (GAS) or Staphylococcus aureus. Analysis of mutants impaired for various surface components revealed that the GBS capsule is a key component in this process.

  2. SOX7 is involved in aspirin-mediated growth inhibition of human colorectal cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xin Zhou; Shu-Yan Huang; Jing-Xin Feng; Yan-Yan Gao; Li Zhao; Jun Lu; Bai-Qu Huang; Yu Zhang

    2011-01-01

    AIM: To confirm the role of sex-determining region Y-box 7 (Sox7) in aspirin-mediated growth inhibition of COX-independent human colorectal cancer cells.METHODS: The cell survival percentage was examined by MTT (Moto-nuclear cell direc cytotoxicity) assay.SOX7 expression was assessed by using reverse transcription-polymerase chain reaction and Western blotting. SB203580 was used to inhibit the p38MAPK signal pathway. SOX7 promoter activity was detected by Luciferase reporter assay.RESULTS: SOX7 was upregulated by aspirin and was involved in aspirin-mediated growth inhibition of SW480 human colorectal cancer cells. The p38MAPK pathway played a role in aspirin-induced SOX7 expression, during which the AP1 transcription factors c-Jun and c-Fos upregulated SOX7 promoter activities.RESULTS: SOX7 is upregulated by aspirin and is involved in aspirin-mediated growth inhibition of human colorectal cancer SW480 cells.

  3. Control of Dog Mediated Human Rabies in Haiti: No Time to Spare.

    Directory of Open Access Journals (Sweden)

    Max F Millien

    Full Text Available The American region has pledged to eliminate dog-mediated human rabies by 2015. As part of these efforts, we describe the findings of a desk and field mission review of Haiti's rabies situation by the end of 2013. While government officials recognize the importance of dog-mediated rabies control, and the national rabies plan adequately contemplates the basic capacities to that effect, regular and sufficient implementation, for example, of dog vaccination, is hampered by limited funding. Compounding insufficient funding and human resources, official surveillance figures do not accurately reflect the risk to the population, as evidenced by the large number of rabid dogs detected by focalized and enhanced surveillance activities conducted by the Ministry of Agriculture, Natural Resources and Rural Development (MARNDR and the Health and Population Ministry (MSPP with the technical assistance of the United States Centers for Disease Control and Prevention. Although international support is common, either in the form of on-the-ground technical support or donations of immunobiologicals, it is not comprehensive. In addition, there is limited coordination with MARNDR/MSPP and with other actors at the strategic or operational level due to human resources limitations. Given these findings, the 2015 elimination goal in the region is compromised by the situation in Haiti where control of the disease is not yet in sight despite the best efforts of the resolute national officials. More importantly, dog-mediated rabies is still a threat to the Haitian population.

  4. Control of Dog Mediated Human Rabies in Haiti: No Time to Spare.

    Science.gov (United States)

    Millien, Max F; Pierre-Louis, Jocelyne B; Wallace, Ryan; Caldas, Eduardo; Rwangabgoba, Jean M; Poncelet, Jean L; Cosivi, Ottorino; Del Rio Vilas, Victor J

    2015-01-01

    The American region has pledged to eliminate dog-mediated human rabies by 2015. As part of these efforts, we describe the findings of a desk and field mission review of Haiti's rabies situation by the end of 2013. While government officials recognize the importance of dog-mediated rabies control, and the national rabies plan adequately contemplates the basic capacities to that effect, regular and sufficient implementation, for example, of dog vaccination, is hampered by limited funding. Compounding insufficient funding and human resources, official surveillance figures do not accurately reflect the risk to the population, as evidenced by the large number of rabid dogs detected by focalized and enhanced surveillance activities conducted by the Ministry of Agriculture, Natural Resources and Rural Development (MARNDR) and the Health and Population Ministry (MSPP) with the technical assistance of the United States Centers for Disease Control and Prevention. Although international support is common, either in the form of on-the-ground technical support or donations of immunobiologicals, it is not comprehensive. In addition, there is limited coordination with MARNDR/MSPP and with other actors at the strategic or operational level due to human resources limitations. Given these findings, the 2015 elimination goal in the region is compromised by the situation in Haiti where control of the disease is not yet in sight despite the best efforts of the resolute national officials. More importantly, dog-mediated rabies is still a threat to the Haitian population.

  5. Modeling nicotinic neuromodulation from global functional and network levels to nAChR based mechanisms

    Institute of Scientific and Technical Information of China (English)

    Michael GRAUPNER; Boris GUTKIN

    2009-01-01

    Neuromodulator action has received increasing attention in theoretical neuroscience. Yet models involving both neuronal populations dynamics at the circuit level and detailed receptor properties are only now being developed. Here we review recent computational approaches to neuromodulation, focusing specifically on acetylcholine (ACh) and nicotine. We dis-cuss illustrative examples of models ranging from functional top-down to neurodynamical bottom-up. In the top-down approach, a computational theory views ACh as encoding the uncertainty expected in an environment. A different line of models accounts for neural population dynamics treating ACh as toggling neuronal networks between read-in of informa-tion and recall of memory. Building on the neurodynamics idea we discuss two models of nicotine's action with increasing degree of biological realism. Both consider explicitly receptor-level mechanisms but with different scales of detail. The first is a large-scale model of nicotine-dependent modulation of dopaminergic signaling that is capable of simulating nicotine self-administration. The second is a novel approach where circuit-level neurodynamics of the ventral tegmental area (VTA) are combined with explicit models of the dynamics of specific nicotinic ACh receptor subtypes. We show how the model is constructed based on local anatomy, electrophysiology and receptor properties and provide an illustration of its potential. In particular, we show how the model can shed light on the specific mechanisms by which nicotine controls dopaminergic neurotransmission in the VTA. This model serves us to conclude that detailed accounts for neuromodulator action at the basis of behavioral and cognitive models are crucial to understand how neuromodulators mediate their functional proper-ties.

  6. Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene

    Science.gov (United States)

    Greenwald, David L.; Cashman, Siobhan M.

    2010-01-01

    Purpose. Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Methods. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Results. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. Conclusions. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination. PMID:20671268

  7. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.

    Science.gov (United States)

    Tang, Lichun; Zeng, Yanting; Du, Hongzi; Gong, Mengmeng; Peng, Jin; Zhang, Buxi; Lei, Ming; Zhao, Fang; Wang, Weihua; Li, Xiaowei; Liu, Jianqiao

    2017-03-01

    Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

  8. The rules and impact of nonsense-mediated mRNA decay in human cancers.

    Science.gov (United States)

    Lindeboom, Rik G H; Supek, Fran; Lehner, Ben

    2016-10-01

    Premature termination codons (PTCs) cause a large proportion of inherited human genetic diseases. PTC-containing transcripts can be degraded by an mRNA surveillance pathway termed nonsense-mediated mRNA decay (NMD). However, the efficiency of NMD varies; it is inefficient when a PTC is located downstream of the last exon junction complex (EJC). We used matched exome and transcriptome data from 9,769 human tumors to systematically elucidate the rules of NMD targeting in human cells. An integrated model incorporating multiple rules beyond the canonical EJC model explains approximately three-fourths of the non-random variance in NMD efficiency across thousands of PTCs. We also show that dosage compensation may sometimes mask the effects of NMD. Applying the NMD model identifies signatures of both positive and negative selection on NMD-triggering mutations in human tumors and provides a classification for tumor-suppressor genes.

  9. Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate

    Directory of Open Access Journals (Sweden)

    Chih-Hong Lou

    2016-06-01

    Full Text Available Nonsense-mediated RNA decay (NMD is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

  10. IMPROVEMENT OF HUMAN ISLET FUNCTION BY ADENOVIRUS MEDIATED HO-1 GENE TRANSFER

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.

  11. Urban dogs in rural areas: Human-mediated movement defines dog populations in southern Chile.

    Science.gov (United States)

    Villatoro, Federico J; Sepúlveda, Maximiliano A; Stowhas, Paulina; Silva-Rodríguez, Eduardo A

    2016-12-01

    Management strategies for dog populations and their diseases include reproductive control, euthanasia and vaccination, among others. However, the effectiveness of these strategies can be severely affected by human-mediated dog movement. If immigration is important, then the location of origin of dogs imported by humans will be fundamental to define the spatial scales over which population management and research should apply. In this context, the main objective of our study was to determine the spatial extent of dog demographic processes in rural areas and the proportion of dogs that could be labeled as immigrants at multiple spatial scales. To address our objective we conducted surveys in households located in a rural landscape in southern Chile. Interviews allowed us to obtain information on the demographic characteristics of dogs in these rural settings, human influence on dog mortality and births, the localities of origin of dogs living in rural areas, and the spatial extent of human-mediated dog movement. We found that most rural dogs (64.1%) were either urban dogs that had been brought to rural areas (40.0%), or adopted dogs that had been previously abandoned in rural roads (24.1%). Some dogs were brought from areas located as far as ∼700km away from the study area. Human-mediated movement of dogs, especially from urban areas, seems to play a fundamental role in the population dynamics of dogs in rural areas. Consequently, local scale efforts to manage dog populations or their diseases are unlikely to succeed if implemented in isolation, simply because dogs can be brought from surrounding urban areas or even distant locations. We suggest that efforts to manage or study dog populations and related diseases should be implemented using a multi-scale approach.

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

    Science.gov (United States)

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

    2015-04-01

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

  13. RAD18 mediates resistance to ionizing radiation in human glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Chen; Wang, Hongwei; Cheng, Hongbin; Li, Jianhua; Wang, Zhi, E-mail: drzwang@gmail.com; Yue, Wu, E-mail: drwuyue@gmail.com

    2014-02-28

    Highlights: • RAD18 is an important mediator of the IR-induced resistance in glioma cell lines. • RAD18 overexpression confers resistance to IR-mediated apoptosis. • The elevated expression of RAD18 is associated with recurrent GBM who underwent IR therapy. - Abstract: Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). RAD18 a central regulator of translesion DNA synthesis (TLS), has been shown to play an important role in regulating genomic stability and DNA damage response. In the present study, we investigate the relationship between RAD18 and resistance to ionizing radiation (IR) and examined the expression levels of RAD18 in primary and recurrent GBM specimens. Our results showed that RAD18 is an important mediator of the IR-induced resistance in GBM. The expression level of RAD18 in glioma cells correlates with their resistance to IR. Ectopic expression of RAD18 in RAD18-low A172 glioma cells confers significant resistance to IR treatment. Conversely, depletion of endogenous RAD18 in RAD18-high glioma cells sensitized these cells to IR treatment. Moreover, RAD18 overexpression confers resistance to IR-mediated apoptosis in RAD18-low A172 glioma cells, whereas cells deficient in RAD18 exhibit increased apoptosis induced by IR. Furthermore, knockdown of RAD18 in RAD18-high glioma cells disrupts HR-mediated repair, resulting in increased accumulation of DSB. In addition, clinical data indicated that RAD18 was significantly higher in recurrent GBM samples that were exposed to IR compared with the corresponding primary GBM samples. Collectively, our findings reveal that RAD18 may serve as a key mediator of the IR response and may function as a potential target for circumventing IR resistance in human GBM.

  14. β-Cell regeneration mediated by human bone marrow mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Anna Milanesi

    Full Text Available Bone marrow mesenchymal stem cells (BMSCs have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into β-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous β-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF or pancreatic-duodenal homeobox 1 (PDX1 to reverse diabetes and whether these cells were differentiated into β-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo β-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous β-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining β-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of β-cell recovery after injury mediated by hBMSC therapy.

  15. Human nicotine conditioning requires explicit contingency knowledge: is addictive behaviour cognitively mediated?

    Science.gov (United States)

    Hogarth, Lee; Duka, Theodora

    2006-03-01

    Two seemingly contrary theories describe the learning mechanisms that mediate human addictive behaviour. According to the classical incentive theories of addiction, addictive behaviour is motivated by a Pavlovian conditioned appetitive emotional response elicited by drug-paired stimuli. Expectancy theory, on the other hand, argues that addictive behaviour is mediated by an expectancy of the drug imparted by cognitive knowledge of the Pavlovian (predictive) contingency between stimuli (S+) and the drug and of the instrumental (causal) contingency between instrumental behaviour and the drug. The present paper reviewed human-nicotine-conditioning studies to assess the role of appetitive emotional conditioning and explicit contingency knowledge in mediating addictive behaviour. The studies reviewed here provided evidence for both the emotional conditioning and the expectancy accounts. The first source of evidence is that nicotine-paired S+ elicit an appetitive emotional conditioned response (CR), albeit only in participants who expect nicotine. Furthermore, the magnitude of this emotional state is modulated by nicotine deprivation/satiation. However, the causal status of the emotional response in driving other forms of conditioned behaviour remains undemonstrated. The second source of evidence is that other nicotine CRs, including physiological responses, self-administration, attentional bias and subjective craving, are also dependent on participants possessing explicit knowledge of the Pavlovian contingencies arranged in the experiment. In addition, several of the nicotine CRs can be brought about or modified by instructed contingency knowledge, demonstrating the causal status of this knowledge. Collectively, these data suggest that human nicotine conditioned effects are mediated by an explicit expectancy of the drug coupled with an appetitive emotional response that reflects the positive biological value of the drug. The implication of this conclusion is that

  16. Comparison of epigenetic mediator expression and function in mouse and human embryonic blastomeres.

    Science.gov (United States)

    Chavez, Shawn L; McElroy, Sohyun L; Bossert, Nancy L; De Jonge, Christopher J; Rodriguez, Maria Vera; Leong, Denise E; Behr, Barry; Westphal, Lynn M; Reijo Pera, Renee A

    2014-09-15

    A map of human embryo development that combines imaging, molecular, genetic and epigenetic data for comparisons to other species and across pathologies would be greatly beneficial for basic science and clinical applications. Here, we compared mRNA and protein expression of key mediators of DNA methylation and histone modifications between mouse and human embryos, embryos from fertile/infertile couples, and following growth factor supplementation. We observed that individual mouse and human embryos are characterized by similarities and distinct differences in DNA methylation and histone modification patterns especially at the single-cell level. In particular, while mouse embryos first exhibited sub-compartmentalization of different histone modifications between blastomeres at the morula stage and cell sub-populations in blastocysts, differential histone modification expression was detected between blastomeres earlier in human embryos at the four- to eight-cell stage. Likewise, differences in epigenetic mediator expression were also observed between embryos from fertile and infertile couples, which were largely equalized in response to growth factor supplementation, suggesting that select growth factors might prevent alterations in epigenetic profiles during prolonged embryo culture. Finally, we determined that reduced expression via morpholino technologies of a single histone-modifying enzyme, Rps6ka4/Msk2, resulted in cleavage-stage arrest as assessed by time-lapse imaging and was associated with aneuploidy generation. Taken together, data document differences in epigenetic patterns between species with implications for fertility and suggest functional roles for individual epigenetic factors during pre-implantation development.

  17. Discovery of Isoxazole Analogs of Sazetidine-A as Selective α4β2-Nicotinic Acetylcholine Receptor (nAChR) Partial Agonists for the Treatment of Depression

    OpenAIRE

    Liu, Jianhua; Yu, Li-Fang; Eaton, J. Brek; Caldarone, Barbara; Cavino, Katie; Ruiz, Christina; Terry, Matthew; Fedolak, Allison; Wang, DaGuang; Ghavami, Afshin; Lowe, David A; Brunner, Dani; Lukas, Ronald J; Kozikowski, Alan P.

    2011-01-01

    Depression, a common neurological condition, is one of the leading causes of disability and suicide worldwide. Standard treatment targeting monoamine transporters selective for the neurotransmitters serotonin and noradrenalin are not able to help many patients that are poor responders. This study advances the development of sazetidine-A analogs that interact with α4β2-nAChR as partial agonists and that possess favorable antidepressant profiles. The resulting compounds that are highly selectiv...

  18. Mediators of compassionate goal intervention effects on human neuroendocrine responses to the Trier Social Stress Test.

    Science.gov (United States)

    Erickson, Thane M; Mayer, Stefanie E; Lopez-Duran, Nestor L; Scarsella, Gina M; McGuire, Adam P; Crocker, Jennifer; Abelson, James L

    2017-08-28

    The hypothalamic-pituitary-adrenal (HPA) axis is thought to mediate the effects of stress on illness. Research has identified a limited number of psychological variables that modulate human HPA responses to stressors (e.g. perceived control and social support). Prosocial goals can reduce subjective stress, but have not been carefully examined in experimental settings where pathways of impact on biological stress markers may be traced. Recent work demonstrated that coaching individuals to strive to help others reduced HPA responses to the Trier Social Stress Test (TSST) relative to other cognitive interventions. However, identification of mediational pathways, which were not examined in the original study, is necessary to determine whether the HPA buffering effects were due to helping motivations (compassionate goals; CGs) rather than via previously identified variables such as control or support. In this new analysis, we combined the original cortisol data with novel observer ratings of interpersonal behavior and psychological variables during the stress task, and conducted new, theory-driven analyses to determine psychological mediators for the intervention's effect on cortisol responses (N = 54; 21 females, 33 males; 486 cortisol samples). Control, support, and task ego-threat failed to account for the effects of the intervention. As hypothesized, self and observer-rated CGs, as well as observer-rated perceptions of participants' interpersonal behavior as morally desirable (but not as dominant or affiliative) were significant mediators of neuroendocrine responses. The findings suggest that stress-reduction interventions based on prosocial behavior should target particular motivational and interpersonal features.

  19. Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells

    Science.gov (United States)

    Hyland, Kendra A.; Olson, Erik R.; McIvor, R. Scott

    2015-01-01

    The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34+ HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon–chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications. PMID:26176276

  20. GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

    Institute of Scientific and Technical Information of China (English)

    Nariman A B Balenga; Maria Waldhoer; Elma Aflaki; Julia Kargl; Wolfgang Platzer; Ralf Schr(o)der; Stefanie Bl(a)ttermann; Evi Kostenis; Andrew J Brown; Akos Heinemann

    2011-01-01

    The directional migration of neutrophils towards inflammatory mediators,such as chemokines and cannabinoids,occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process.A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB2 receptor (CB2R),but additional modulatory sites distinct from CB2R have recently been suggested to impact CB2R-mediated effector functions in neutrophils.Here,we provide evidence that the recently de-orphanized 7TM/GPCR GPR55potently modulates CB2R-mediated responses.We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB2R agonist 2-arachidonoylglycerol (2-AG),while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production.Using HEK293 and HL60 cell lines,along with primary neutrophils,we show that GPR55 and CB2R interfere with each other's signaling pathways at the level of small GTPases,such as Rac2 and Cdc42.This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils.Therefore,GPR55 limits the tissueinjuring inflammatory responses mediated by CB2R,while it synergizes with CB2R in recruiting neutrophils to sites of inflammation.

  1. The importance of human FcgammaRI in mediating protection to malaria.

    Directory of Open Access Journals (Sweden)

    Richard S McIntosh

    2007-05-01

    Full Text Available The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.

  2. Quantitation of the Contractile Response Mediated by Two Receptors: M2 and M3 Muscarinic Receptor-Mediated Contractions of Human Gastroesophageal Smooth MuscleS⃞

    Science.gov (United States)

    Braverman, Alan S.; Miller, Larry S.; Vegesna, Anil K.; Tiwana, Mansoor I.; Tallarida, Ronald J.; Ruggieri, Michael R.

    2009-01-01

    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M2 and M3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M2 and M3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M2 and M3 receptors to contraction is presented. Using this new method of analysis, it was found that the M2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction. PMID:19126780

  3. Quantitation of the contractile response mediated by two receptors: M2 and M3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle.

    Science.gov (United States)

    Braverman, Alan S; Miller, Larry S; Vegesna, Anil K; Tiwana, Mansoor I; Tallarida, Ronald J; Ruggieri, Michael R

    2009-04-01

    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M(2) and M(3) muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M(2) receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M(3) density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M(2) and M(3) receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M(2) and M(3) receptors to contraction is presented. Using this new method of analysis, it was found that the M(2) muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M(3) receptor predominantly mediates contraction.

  4. Axl Mediates ZIKA Virus Entry in Human Glial Cells and Modulates Innate Immune Responses

    Directory of Open Access Journals (Sweden)

    Laurent Meertens

    2017-01-01

    Full Text Available ZIKA virus (ZIKV is an emerging pathogen responsible for neurological disorders and congenital microcephaly. However, the molecular basis for ZIKV neurotropism remains poorly understood. Here, we show that Axl is expressed in human microglia and astrocytes in the developing brain and that it mediates ZIKV infection of glial cells. Axl-mediated ZIKV entry requires the Axl ligand Gas6, which bridges ZIKV particles to glial cells. Following binding, ZIKV is internalized through clathrin-mediated endocytosis and traffics to Rab5+ endosomes to establish productive infection. During entry, the ZIKV/Gas6 complex activates Axl kinase activity, which downmodulates interferon signaling and facilitates infection. ZIKV infection of human glial cells is inhibited by MYD1, an engineered Axl decoy receptor, and by the Axl kinase inhibitor R428. Our results highlight the dual role of Axl during ZIKV infection of glial cells: promoting viral entry and modulating innate immune responses. Therefore, inhibiting Axl function may represent a potential target for future antiviral therapies.

  5. TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells.

    Science.gov (United States)

    Mitrovic, Sandra; Nogueira, Cristina; Cantero-Recasens, Gerard; Kiefer, Kerstin; Fernández-Fernández, José M; Popoff, Jean-François; Casano, Laetitia; Bard, Frederic A; Gomez, Raul; Valverde, Miguel A; Malhotra, Vivek

    2013-05-28

    Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion. DOI:http://dx.doi.org/10.7554/eLife.00658.001.

  6. Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and"Death factor"family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of"Death factor" family, the transfection experiments with expression vectors pcDNA3-fland pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl2. The data showed that the expression of FasL in pcDNA3fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fltransient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hcpatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.

  7. Induction of long-term oscillations in the γ frequency band by nAChR activation in rat hippocampal CA3 area.

    Science.gov (United States)

    Zhang, X; Ge, X Y; Wang, J G; Wang, Y L; Wang, Y; Yu, Y; Li, P P; Lu, C B

    2015-08-20

    The hippocampal neuronal network oscillation at γ frequency band (γ oscillation) is generated by the precise interaction between interneurons and principle cells. γ oscillation is associated with attention, learning and memory and is impaired in the diseased conditions such as Alzheimer's disease (AD) and schizophrenia. Nicotinic acetylcholine receptor (nAChR) plays an important role in the regulation of hippocampal neurotransmission and network activity. It is not known whether nicotine modulates plasticity of network activity at γ oscillations in the hippocampus. In this study we investigated the effects of nicotine on the long-term changes of KA-induced γ oscillations. We found that hippocampal γ oscillations can be enhanced by a low concentration of nicotine (1μM), such an enhancement lasts for hours after washing out of nicotine, suggesting a form of synaptic plasticity, named as long-term oscillation at γ frequency band (LTOγ). Nicotine-induced LTOγ was mimicked by the selective α4β2 but not by α7 nAChR agonist and was involved in N-methyl-d-aspartate (NMDA) receptor activation as well as depended on excitatory and inhibitory neurotransmission. Our results indicate that nAChR activation induced plasticity in γ oscillation, which may be beneficial for the improvement of cognitive deficiency in AD and schizophrenia.

  8. Impairment of contextual fear extinction by chronic nicotine and withdrawal from chronic nicotine is associated with hippocampal nAChR upregulation.

    Science.gov (United States)

    Kutlu, Munir Gunes; Oliver, Chicora; Huang, Peng; Liu-Chen, Lee-Yuan; Gould, Thomas J

    2016-10-01

    Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of fear learning behavior. Moreover, recent studies from our laboratory have shown that acute nicotine impaired fear extinction and safety learning in mice. However, the effects of chronic nicotine and withdrawal on fear extinction are unknown. Therefore, the current experiments were conducted to investigate the effects of chronic nicotine as well as withdrawal from chronic nicotine on contextual fear extinction in mice. C57BL6/J mice were given contextual fear conditioning training and retention testing during chronic nicotine administration. Mice then received contextual fear extinction either during chronic nicotine or during withdrawal from chronic nicotine. Our results showed that contextual fear extinction was impaired both during chronic nicotine administration and subsequent withdrawal. However, it was also observed that the effects of prior chronic nicotine disappeared after 72 h in withdrawal, a timeline that closely matches with the timing of the chronic nicotine-induced upregulation of hippocampal nicotinic acetylcholine receptor (nAChR) density. Additional experiments found that 4 days, but not 1 day, of continuous nicotine administration upregulated hippocampal nAChRs and impaired contextual fear extinction. These effects disappeared following 72 h withdrawal. Overall, these experiments provide a potential link between nicotine-induced upregulation of hippocampal nAChRs and fear extinction deficits observed in patients with anxiety disorders, which may lead to advancements in the pharmacological treatment methods for this disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Inhibition of copper-mediated aggregation of human γD-crystallin by Schiff bases.

    Science.gov (United States)

    Chauhan, Priyanka; Muralidharan, Sai Brinda; Velappan, Anand Babu; Datta, Dhrubajyoti; Pratihar, Sanjay; Debnath, Joy; Ghosh, Kalyan Sundar

    2017-01-05

    Protein aggregation, due to the imbalance in the concentration of Cu(2+) and Zn(2+) ions is found to be allied with various physiological disorders. Copper is known to promote the oxidative damage of β/γ-crystallins in aged eye lens and causes their aggregation leading to cataract. Therefore, synthesis of a small-molecule 'chelator' for Cu(2+) with complementary antioxidant effect will find potential applications against aggregation of β/γ-crystallins. In this paper, we have reported the synthesis of different Schiff bases and studied their Cu(2+) complexation ability (using UV-Vis, FT-IR and ESI-MS) and antioxidant activity. Further based on their copper complexation efficiency, Schiff bases were used to inhibit Cu(2+)-mediated aggregation of recombinant human γD-crystallin (HGD) and β/γ-crystallins (isolated from cataractous human eye lens). Among these synthesized molecules, compound 8 at a concentration of 100 μM had shown ~95% inhibition of copper (100 μM)-induced aggregation. Compound 8 also showed a positive cooperative effect at a concentration of 5-15 μM on the inhibitory activity of human αA-crystallin (HAA) during Cu(2+)-induced aggregation of HGD. It eventually inhibited the aggregation process by additional ~20%. However, ~50% inhibition of copper-mediated aggregation of β/γ-crystallins (isolated from cataractous human eye lens) was recorded by compound 8 (100 μM). Although the reductive aminated products of the imines showed better antioxidant activity due to their lower copper complexing ability, they were found to be non-effective against Cu(2+)-mediated aggregation of HGD.

  10. P2X7 receptors mediate innate phagocytosis by human neural precursor cells and neuroblasts.

    Science.gov (United States)

    Lovelace, Michael D; Gu, Ben J; Eamegdool, Steven S; Weible, Michael W; Wiley, James S; Allen, David G; Chan-Ling, Tailoi

    2015-02-01

    During early human neurogenesis there is overproduction of neuroblasts and neurons accompanied by widespread programmed cell death (PCD). While it is understood that CD68(+) microglia and astrocytes mediate phagocytosis during target-dependent PCD, little is known of the cell identity or the scavenger molecules used to remove apoptotic corpses during the earliest stages of human neurogenesis. Using a combination of multiple-marker immunohistochemical staining, functional blocking antibodies and antagonists, we showed that human neural precursor cells (hNPCs) and neuroblasts express functional P2X7 receptors. Furthermore, using live-cell imaging, flow cytometry, phagocytic assays, and siRNA knockdown, we showed that in a serum-free environment, doublecortin(+) (DCX) neuroblasts and hNPCs can clear apoptotic cells by innate phagocytosis mediated via P2X7. We found that both P2X7(high) DCX(low) hNPCs and P2X7(high) DCX(high) neuroblasts, derived from primary cultures of human fetal telencephalon, phagocytosed targets including latex beads, apoptotic ReNcells, and apoptotic hNPC/neuroblasts. Pretreatment of neuroblasts and hNPCs with 1 mM adenosine triphosphate (ATP), 100 µM OxATP (P2X7 antagonist), or siRNA knockdown of P2X7 inhibited phagocytosis of these targets. Our results show that P2X7 functions as a scavenger receptor under serum-free conditions resembling those in early neurogenesis. This is the first demonstration that hNPCs and neuroblasts may participate in clearance of apoptotic corpses during pre target-dependent neurogenesis and mediate phagocytosis using P2X7 as a scavenger receptor.

  11. Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes.

    Science.gov (United States)

    Gao, Dan; Madi, Mohamed; Ding, Cherlyn; Fok, Matthew; Steele, Thomas; Ford, Christopher; Hunter, Leif; Bing, Chen

    2014-08-01

    Adipose tissue expansion during obesity is associated with increased macrophage infiltration. Macrophage-derived factors significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. Identification of the major factors that mediate detrimental effects of macrophages on adipocytes may offer potential therapeutic targets. IL-1β, a proinflammatory cytokine, is suggested to be involved in the development of insulin resistance. This study investigated the role of IL-1β in macrophage-adipocyte cross-talk, which affects insulin signaling in human adipocytes. Using macrophage-conditioned (MC) medium and human primary adipocytes, we examined the effect of IL-1β antagonism on the insulin signaling pathway. Gene expression profile and protein abundance of insulin signaling molecules were determined, as was the production of proinflammatory cytokine/chemokines. We also examined whether IL-1β mediates MC medium-induced alteration in adipocyte lipid storage. MC medium and IL-1β significantly reduced gene expression and protein abundance of insulin signaling molecules, including insulin receptor substrate-1, phosphoinositide 3-kinase p85α, and glucose transporter 4 and phosphorylation of Akt. In contrast, the expression and release of the proinflammatory markers, including IL-6, IL-8, monocyte chemotactic protein-1, and chemokine (C-C motif) ligand 5 by adipocytes were markedly increased. These changes were significantly reduced by blocking IL-1β activity, its receptor binding, or its production by macrophages. MC medium-inhibited expression of the adipogenic factors and -stimulated lipolysis was also blunted with IL-1β neutralization. We conclude that IL-1β mediates, at least in part, the effect of macrophages on insulin signaling and proinflammatory response in human adipocytes. Blocking IL-1β could be beneficial for preventing obesity-associated insulin resistance and inflammation in human adipose tissue. Copyright

  12. Human macrophage foam cells degrade atherosclerotic plaques through cathepsin K mediated processes

    DEFF Research Database (Denmark)

    Barascuk, Natasha; Skjøt-Arkil, Helene; Register, Thomas C

    2010-01-01

    BACKGROUND: Proteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture.The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular......-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in women...... with aortic calcifications compared to those without. CONCLUSIONS: Human macrophage foam cells degrade the atherosclerotic plaques though cathepsin K mediated processes, resulting in increase in levels of CTX-I. Serum CTX-I was not elevated in women with aortic calcification, likely due to the contribution...

  13. Loss of the tumour suppressor gene AIP mediates the browning of human brown fat tumours.

    Science.gov (United States)

    Magnusson, Linda; Hansen, Nils; Saba, Karim H; Nilsson, Jenny; Fioretos, Thoas; Rissler, Pehr; Nord, Karolin H

    2017-10-01

    Human brown fat tumours (hibernomas) show concomitant loss of the tumour suppressor genes MEN1 and AIP. We hypothesized that the brown fat phenotype is attributable to these mutations. Accordingly, in this study, we demonstrate that silencing of AIP in human brown preadipocytic and white fat cell lines results in the induction of the brown fat marker UCP1. In human adipocytic tumours, loss of MEN1 was found both in white (one of 51 lipomas) and in brown fat tumours. In contrast, concurrent loss of AIP was always accompanied by a brown fat morphology. We conclude that this white-to-brown phenotype switch in brown fat tumours is mediated by the loss of AIP. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  14. Human macrophage foam cells degrade atherosclerotic plaques through cathepsin K mediated processes

    Directory of Open Access Journals (Sweden)

    Larsen Lise

    2010-04-01

    Full Text Available Abstract Background Proteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture. The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular matrix (ECM of atherosclerotic plaques by cathepsin K mediated processes. Methods We 1 cultured human macrophages on ECM and measured cathepsin K generated fragments of type I collagen (C-terminal fragments of Type I collagen (CTX-I 2 investigated the presence of CTX-I in human coronary arteries and 3 finally investigated the clinical potential by measuring circulating CTX-I in women with and without radiographic evidence of aortic calcified atherosclerosis. Results Immune-histochemistry of early and advanced lesions of coronary arteries demonstrated co-localization of Cathepsin-K and CTX-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in women with aortic calcifications compared to those without. Conclusions Human macrophage foam cells degrade the atherosclerotic plaques though cathepsin K mediated processes, resulting in increase in levels of CTX-I. Serum CTX-I was not elevated in women with aortic calcification, likely due to the contribution of CTX-I from osteoclastic bone resorption which involves Cathepsin-K. The human macrophage model system may be used to identify important pathway leading to excessive proteolytic plaque remodeling and plaque rupture.

  15. DOES PERCEIVED ORGANIZATIONAL SUPPORT MEDIATE THE RELATIONSHIP BETWEEN HUMAN RESOURCE MANAGEMENT PRACTICES AND ORGANIZATIONAL COMMITMENT?

    Directory of Open Access Journals (Sweden)

    Aizzat Mohd. Nasurdin

    2008-01-01

    Full Text Available This study examines a model involving Human Resource Management (HRM practices, perceived organizational support, and organizational commitment. It was hypothesized that HRM practices (performance appraisal, training and career development will be positively related to organizational commitment, and that perceived organizational support would serve as a mediator in the relationship between HRM practices and commitment. The statistical results on data gathered from a sample of 214 employees within the Malaysian manufacturing sector demonstrated that career development and performance appraisal have direct, positive and significant relationships with organizational commitment. In addition, perceived organizational support was found to partially mediate the relationships between two of the three HRM practices (career development and performance appraisal and commitment. Theoretical and managerial implications are suggested.

  16. MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro.

    Science.gov (United States)

    Khodadadian, M; Leroux, M E; Auzenne, E; Ghosh, S C; Farquhar, D; Evans, R; Spohn, W; Zou, Y; Klostergaard, J

    2009-10-01

    Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

  17. Phenobarbital-mediated tumor promotion in transgenic mice with humanized CAR and PXR.

    Science.gov (United States)

    Braeuning, Albert; Gavrilov, Alina; Brown, Susan; Wolf, C Roland; Henderson, Colin J; Schwarz, Michael

    2014-08-01

    The nuclear receptors CAR (constitutive androstane receptor) and possibly PXR (pregnane X receptor) mediate the hepatic effects of phenobarbital (PB) and similar-acting compounds. Although PB is a potent nongenotoxic tumor promoter in rodent liver, epidemiological data from epilepsy patients treated with phenobarbital do not show a specific role of PB in human liver cancer risk. That points to species differences in the susceptibility to tumor promotion by PB, which might be attributed to divergent functions of the PB receptors CAR and PXR in mice and humans. In the present study, male transgenic mice expressing human CAR and PXR were used to detect possible differences between wild-type (WT) and humanized mice in their response to CAR activation in a tumor initiation/promotion experiment with a single injection of the tumor initiator N-nitrosodiethylamine preceding chronic PB treatment for 10 months. Analysis of liver tumor burden revealed that PB strongly promoted the outgrowth of hepatocellular adenoma driven by activated β-catenin in WT mice, whereas the tumor-promoting effect of PB was much less pronounced in the humanized group. In conclusion, the present findings demonstrate that human CAR and PXR support tumor promotion by PB in mouse liver, but to a significantly lesser extent than the WT murine receptors. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Combined postconditioning with ischemia and α7nAChR agonist produces an enhanced protection against rat myocardial ischemia reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    XIONG Jun; YUAN Yu-jing; XUE Fu-shan; WANG Qiang; LI Shan; LIAO Xu; LIU Jian-hua; CHEN Yi; LI Rui-ping

    2012-01-01

    Background Inflammation is one of important mechanisms for myocardial ischemia reperfusion injury (IRI).Ischemia postconditioning (IPOC) can protect the heart against IRI by inhibiting inflammation,but its cardioprotection is weaker than that of ischemia preconditioning.Recently,the α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) agonist has shown anti-infiammatory effects in many diseases related to inflammation.This randomized controlled experiment was designed to evaluate whether combined postconditioning with IPOC and the α7nAChR agonist could produce an enhanced cardioprotection in a rat in vivo model of acute myocardial IRI.Methods Fifty Sprague-Dawley rats were randomly divided into five equal groups:sham group,control group,IPOC group,α7nAChR agonist postconditioning group (APOC group) and combined postconditioning with IPOC and α7nAChR agonist group (combined group).Hemodynamic parameters were recorded during the periods of ischemia and reperfusion.Serum concentrations of troponin I (Tnl),tumor necrosis factor α (TNF-α) and high-mobility group box 1 (HMGB-1) at 180 minutes after reperfusion were assayed in all groups.At the end of the experiment,the infarct size was assessed from excised hearts by Evans blue and triphenyl tetrazolium chloride staining.Results As compared to the sham group,the infarct size in the other four groups was significantly increased,serum levels of Tnl,TNF-α and HMGB1 in the control group and TNF-α,HMGB1 in the IPOC group were significantly increased.The infarct size and serum concentrations of TNF-α,HMGB1 and Tnl in the IPOC,APOC and combined groups were significantly lower than those in the control group.As compared to the IPOC group,the infarct size in the combined group was significantly decreased,serum concentrations of Tnl,TNF-α and HMGB1 in the APOC and combined groups were significantly reduced.Although the infarct size was significantly smaller in the combined group than in the APOC group

  19. Suppression of Human Tenon Fibroblast Cell Proliferation by Lentivirus-Mediated VEGF Small Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Zhongqiu Li

    2017-01-01

    Full Text Available Purpose. The functions of vascular endothelial growth factor (VEGF in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery.

  20. Suppression of Human Tenon Fibroblast Cell Proliferation by Lentivirus-Mediated VEGF Small Hairpin RNA.

    Science.gov (United States)

    Li, Zhongqiu; Hua, Wen; Li, Xuedong; Wang, Wei

    2017-01-01

    Purpose. The functions of vascular endothelial growth factor (VEGF) in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF) cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF) were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery.

  1. IL-17A mediates inflammatory and tissue remodelling events in early human tendinopathy

    Science.gov (United States)

    Millar, Neal L.; Akbar, Moeed; Campbell, Abigail L.; Reilly, James H.; Kerr, Shauna C.; McLean, Michael; Frleta-Gilchrist, Marina; Fazzi, Umberto G.; Leach, William J.; Rooney, Brian P.; Crowe, Lindsay A. N.; Murrell, George A. C.; McInnes, Iain B.

    2016-01-01

    Increasingly, inflammatory mediators are considered crucial to the onset and perpetuation of tendinopathy. We sought evidence of interleukin 17A (IL-17A) expression in early human tendinopathy and thereafter, explored mechanisms whereby IL-17A mediated inflammation and tissue remodeling in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) along with control biopsies were collected from patients undergoing shoulder surgery. Markers of inflammation and IL-17A were quantified by RT-PCR and immunohistochemistry. Human tendon cells were derived from hamstring tendon obtained during ACL reconstruction. In vitro effects of IL-17A upon tenocytes were measured using RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Increased expression of IL-17A was detected in ‘early tendinopathy’ compared to both matched samples and non-matched control samples (p tendinopathy processes thus providing novel therapeutic approaches in the management of tendon disorders. PMID:27263531

  2. XB130 translocation to microfilamentous structures mediates NNK-induced migration of human bronchial epithelial cells.

    Science.gov (United States)

    Wu, Qifei; Nadesalingam, Jeya; Moodley, Serisha; Bai, Xiaohui; Liu, Mingyao

    2015-07-20

    Cigarette smoking contributes to the pathogenesis of chronic obstructive pulmonary disease and lung cancer. Nicotine-derived nitrosamine ketone (NNK) is the most potent carcinogen among cigarette smoking components, and is known to enhance migration of cancer cells. However, the effect of NNK on normal human bronchial epithelial cells is not well studied. XB130 is a member of actin filament associated protein family and is involved in cell morphology changes, cytoskeletal rearrangement and outgrowth formation, as well as cell migration. We hypothesized that XB130 mediates NNK-induced migration of normal human bronchial epithelial cells. Our results showed that, after NNK stimulation, XB130 was translocated to the cell periphery and enriched in cell motility-associated structures, such as lamellipodia, in normal human bronchial epithelial BEAS2B cells. Moreover, overexpression of XB130 significantly enhanced NNK-induced migration, which requires both the N- and C-termini of XB130. Overexpression of XB130 enhanced NNK-induced protein tyrosine phosphorylation and promoted matrix metalloproteinase-14 translocation to cell motility-associated cellular structures after NNK stimulation. XB130-mediated NNK-induced cell migration may contribute to airway epithelial repair; however, it may also be involved in cigarette smoking-related chronic obstructive pulmonary disease and lung cancer.

  3. HMGB1-mediated autophagy decreases sensitivity to oxymatrine in SW982 human synovial sarcoma cells.

    Science.gov (United States)

    Cai, Yongsong; Xu, Peng; Yang, Le; Xu, Ke; Zhu, Jialin; Wu, Xiaoqing; Jiang, Congshan; Yuan, Qiling; Wang, Bo; Li, Yuanbo; Qiu, Yusheng

    2016-11-29

    Oxymatrine (OMT) is a type of alkaloid extracted from a traditional Chinese medicinal herb, Sophora flavescens. Although the antitumor activities of OMT have been observed in various cancers, there are no reports regarding the effects of OMT on human synovial sarcoma. In the present study, we analyzed the antitumor activities of OMT in SW982 human synovial sarcoma cells and determine whether high mobility group box protein 1 (HMGB1)-mediated autophagy was associated with its therapeutic effects. We found that OMT exhibited antitumor activity in SW982 cells and facilitated increases in autophagy. Inhibition of autophagy by 3-MA or ATG7 siRNA increased the level of apoptosis, which indicated that OMT-induced autophagy protected cells from the cytotoxicity of OMT. Administration of OMT to SW982 cells increased the expression of HMGB1. When HMGB1 was inhibited via HMGB1-siRNA, OMT-induced autophagy was decreased, and apoptosis was increased. Furthermore, we found that HMGB1-siRNA significantly increased the expression of p-Akt and p-mTOR. OMT-induced autophagy may be mediated by the Akt/mTOR pathway, and HMGB1 plays a vital role in the regulation of autophagy. Therefore, we believe that combining OMT with an inhibitor of autophagy or HMGB1 may make OMT more effective in the treatment of human synovial sarcoma.

  4. STAT4-mediated transcriptional repression of the IL5 gene in human memory Th2 cells.

    Science.gov (United States)

    Gonzales-van Horn, Sarah R; Estrada, Leonardo D; van Oers, Nicolai S C; Farrar, J David

    2016-06-01

    Type I interferon (IFN-α/β) plays a critical role in suppressing viral replication by driving the transcription of hundreds of interferon-sensitive genes (ISGs). While many ISGs are transcriptionally activated by the ISGF3 complex, the significance of other signaling intermediates in IFN-α/β-mediated gene regulation remains elusive, particularly in rare cases of gene silencing. In human Th2 cells, IFN-α/β signaling suppressed IL5 and IL13 mRNA expression during recall responses to T-cell receptor (TCR) activation. This suppression occurred through a rapid reduction in the rate of nascent transcription, independent of de novo expression of ISGs. Further, IFN-α/β-mediated STAT4 activation was required for repressing the human IL5 gene, and disrupting STAT4 dimerization reversed this effect. This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN-α/β signaling and highlights the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells.

  5. Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

    Directory of Open Access Journals (Sweden)

    Nathalie Swales

    Full Text Available AIMS/HYPOTHESIS: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3. In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. METHODS: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. RESULTS: Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. CONCLUSIONS/INTERPRETATION: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

  6. The experimental study of genetic engineering human neural stem cells mediated by lentivirus to express multigene

    Institute of Scientific and Technical Information of China (English)

    CAI Pei-qiang; TANG Xun; LIN Yue-qiu; Oudega Martin; SUN Guang-yun; XU Lin; YANG Yun-kang; ZHOU Tian-hua

    2006-01-01

    Objective:To explore the feasibility to construct genetic engineering human neural stem cells (hNSCs)mediated by lentivirus to express multigene in order to provide a graft source for further studies of spinal cord injury (SCI).Methods: Human neural stem cells from the brain cortex of human abortus were isolated and cultured, then gene was modified by lentivirus to express both green fluorescence protein (GFP) and rat neurotrophin-3(NT-3); the transgenic expression was detected by the methods of fluorescence microscope, dorsal root ganglion of fetal rats and slot blot.Results: Genetic engineering hNSCs were successfully constructed. All of the genetic engineering hNSCs which expressed bright green fluorescence were observed under the fluorescence microscope. The conditioned medium of transgenic hNSCs could induce neurite flourishing outgrowth from dorsal root ganglion (DRG). The genetic engineering hNSCs expressed high level NT-3 which could be detected by using slot blot.Conclusions: Genetic engineering hNSCs mediated by lentivirus can be constructed to express multigene successfully.

  7. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Zwart, Ruud; Ursu, Daniel;

    2015-01-01

    AChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293...... cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology...

  8. MicroRNA-30b-mediated regulation of catalase expression in human ARPE-19 cells.

    Directory of Open Access Journals (Sweden)

    Rashidul Haque

    Full Text Available BACKGROUND: Oxidative injury to retinal pigment epithelium (RPE and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD. Reactive oxygen species (ROS-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19 that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H(2O(2 radicals. Exposure to several stress-inducing agents including H(2O(2 has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H(2O(2 (200 µM up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment. CONCLUSIONS/SIGNIFICANCE: We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

  9. Apelin mediates the induction of profibrogenic genes in human hepatic stellate cells.

    Science.gov (United States)

    Melgar-Lesmes, Pedro; Casals, Gregori; Pauta, Montserrat; Ros, Josefa; Reichenbach, Vedrana; Bataller, Ramon; Morales-Ruiz, Manuel; Jimenez, Wladimiro

    2010-11-01

    Apelin is a peptide with relevant functions in angiogenesis and inflammation. Recent studies have demonstrated that apelin is overexpressed in hepatic stellate cells (HSCs) of cirrhotic rats. Moreover, patients with cirrhosis show high circulating levels of this peptide. We evaluated the role of endogenous apelin system in fibrogenesis-related gene induction in human HSCs. Messenger expression and immunolocalization of apelin were analyzed in human cirrhotic liver and in control samples. Apelin expression was analyzed in a human HSC line (LX-2) under hypoxic conditions or in the presence of proinflammatory or profibrogenic stimuli. LX-2 cells were stimulated with apelin, and a selected profile of fibrogenesis-related genes was quantified. In vivo inactivation of apelin was analyzed in the liver of fibrotic rats after administrating specific blockers of the molecules triggering apelin induction. Apelin was overexpressed in HSCs from human cirrhotic liver. Neither hypoxia nor proinflammatory substances induced the expression of apelin in LX-2. By contrast, both profibrogenic molecules angiotensin II (AII) and endothelin-1 (ET-1) enhanced apelin expression in these cells. Apelin increased the synthesis of collagen-I and platelet-derived growth factor receptor β (PDGFRβ) in LX-2. AII and ET-1 stimulated collagen-I and PDGFRβ expression, and this induction was drastically reduced when apelin receptor was blocked in these cells. In accordance, AII or ET-1 receptor antagonists reduced the hepatic synthesis of apelin, collagen-I, and PDGFRβ in fibrotic rats. apelin mediates some of the fibrogenic effects triggered by AII and ET-1, thus suggesting that apelin could be an important mediator of fibrogenesis in human liver disease.

  10. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    Science.gov (United States)

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  11. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    OpenAIRE

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-01-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its o...

  12. UNEARTHING GLOBAL FINANCIAL INCLUSION LEVELS AND ANALYSIS OF FINANCIAL INCLUSION AS A MEDIATING FACTOR IN GLOBAL HUMAN DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Roshny Unnikrishnan

    2015-04-01

    Full Text Available This study is a result of the author’s inquisition to unearth the current values of Global Financial Inclusion and its relationship with economic growth measured by Gross Domestic product(GDP and human development measured by United Nations Human Development Index (HDI. The Financial Inclusion (FI levels are measured using Index for Financial Inclusion .The relationship between GDP and HDI with FI as mediator, using multiple regression, is validated on a global level based on data of 162 countries for the year 2011. An overall global mediation analysis is undertaken to establish Financial Inclusion as a mediating factor and partial mediation on human development is validated. The study is valid and unique in the global context of income inequality prevailing in developed, developing and underdeveloped countries as it validates the argument that an impressive GDP performance does not ensure equity in economic growth.

  13. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

    Science.gov (United States)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

    1989-06-01

    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  14. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Institute of Scientific and Technical Information of China (English)

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

  15. Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages.

    Science.gov (United States)

    Zhang, Yumei; Pan, Yu; Bian, Zhixiang; Chen, Peihua; Zhu, Shijian; Gu, Huiyi; Guo, Liping; Hu, Chun

    2016-01-01

    Here, we studied the underlying mechanism of aldosterone (Aldo)-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L) inhibited human umbilical vein endothelial cells (HUVEC) survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18) production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P), an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS) inhibitor PDMP or the ceramide (C6) potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR) antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1) is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.

  16. Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC Damages.

    Directory of Open Access Journals (Sweden)

    Yumei Zhang

    Full Text Available Here, we studied the underlying mechanism of aldosterone (Aldo-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L inhibited human umbilical vein endothelial cells (HUVEC survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18 production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P, an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS inhibitor PDMP or the ceramide (C6 potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1 is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.

  17. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    Science.gov (United States)

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  18. Tensile force transmission in human patellar tendon fascicles is not mediated by glycosaminoglycans

    DEFF Research Database (Denmark)

    Svensson, René B; Hassenkam, Tue; Hansen, Philip;

    2011-01-01

    change the tendon modulus, relative energy dissipation, peak stress, or peak strain. The effect of deformation rate was not modulated by the treatment either, indicating no effect on viscosity. These results suggest that GAGs cannot be considered mediators of tensile force transmission in the human...... the mechanical properties of fascicles from human patellar tendon at two different deformation rates before and after removal of GAGs by treatment with chondroitinase ABC. Efficiency of enzyme treatment was quantified using dimethylmethylene blue assay. Removal of at least 79% of the GAGs did not significantly...... patellar tendon, and as such, force transmission must either take place through other matrix components or the fibrils must be mechanically continuous at least to the tested length of 7 mm....

  19. The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system.

    Science.gov (United States)

    Molina-Cruz, Alvaro; Garver, Lindsey S; Alabaster, Amy; Bangiolo, Lois; Haile, Ashley; Winikor, Jared; Ortega, Corrie; van Schaijk, Ben C L; Sauerwein, Robert W; Taylor-Salmon, Emma; Barillas-Mury, Carolina

    2013-05-24

    Plasmodium falciparum transmission by Anopheles gambiae mosquitoes is remarkably efficient, resulting in a very high prevalence of human malaria infection in sub-Saharan Africa. A combination of genetic mapping, linkage group selection, and functional genomics was used to identify Pfs47 as a P. falciparum gene that allows the parasite to infect A. gambiae without activating the mosquito immune system. Disruption of Pfs47 greatly reduced parasite survival in the mosquito, and this phenotype could be reverted by genetic complementation of the parasite or by disruption of the mosquito complement-like system. Pfs47 suppresses midgut nitration responses that are critical to activate the complement-like system. We provide direct experimental evidence that immune evasion mediated by Pfs47 is critical for efficient human malaria transmission by A. gambiae.

  20. 1,25 Dihydroxyvitamin D3 Inhibits TGFβ1-Mediated Primary Human Cardiac Myofibroblast Activation.

    Directory of Open Access Journals (Sweden)

    Anna Meredith

    Full Text Available Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av, and investigated the relationship between circulating vitamin D (25(OHD3 and cardiac fibrosis in human myocardial samples.Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OHD3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGFβ1 to induce activation, in the presence or absence of active vitamin D (1,25(OH2D3. Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH2D3. Treatment with 1,25(OH2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.

  1. Piperine inhibits IL-β induced expression of inflammatory mediators in human osteoarthritis chondrocyte.

    Science.gov (United States)

    Ying, Xiaozhou; Chen, Xiaowei; Cheng, Shaowen; Shen, Yue; Peng, Lei; Xu, Hua Zi

    2013-10-01

    Black pepper (Piper nigrum) is a common remedy in Traditional Chinese Medicine and possesses diverse biological activities including anti-inflammatory properties. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. The present study aimed to assess the effects of piperine, the active phenolic component in black pepper extract, on human OA chondrocytes. In this study, human OA chondrocytes were pretreated with piperine at 10, 50 or 100μg/ml and subsequently stimulated with IL-1β (5ng/ml) for 24h. Production of PGE2 and NO was evaluated by the Griess reaction and an ELISA. Gene expression of MMP-3, MMP-13, iNOS and COX-2 was measured by real-time PCR. MMP-3 and MMP-13 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the iNOS and COX-2 protein production in the culture medium. The regulation of NF-kB activity and the degradation of IkB were explored using luciferase and Western immunoblotting, respectively. We found that piperine inhibited the production of PGE2 and NO induced by IL-1β. Piperine significantly decreased the IL-1β-stimulated gene expression and production of MMP-3, MMP-13, iNOS and COX-2 in human OA chondrocytes. Piperine inhibited the IL-1β-mediated activation of NF-κB by suppressing the degradation of its inhibitory protein IκBα in the cytoplasm. The present report is first to demonstrate the anti-inflammatory activity of piperine in human OA chondrocytes. Piperine can effectively abrogate the IL-1β-induced over-expression of inflammatory mediators; suggesting that piperine may be a potential agent in the treatment of OA.

  2. Role of human-mediated dispersal in the spread of the pinewood nematode in China.

    Directory of Open Access Journals (Sweden)

    Christelle Robinet

    Full Text Available BACKGROUND: Intensification of world trade is responsible for an increase in the number of alien species introductions. Human-mediated dispersal promotes not only introductions but also expansion of the species distribution via long-distance dispersal. Thus, understanding the role of anthropogenic pathways in the spread of invading species has become one of the most important challenges nowadays. METHODOLOGY/PRINCIPAL FINDINGS: We analysed the invasion pattern of the pinewood nematode in China based on invasion data from 1982 to 2005 and monitoring data on 7 locations over 15 years. Short distance spread mediated by long-horned beetles was estimated at 7.5 km per year. Infested sites located further away represented more than 90% of observations and the mean long distance spread was estimated at 111-339 km. Railways, river ports, and lakes had significant effects on the spread pattern. Human population density levels explained 87% of the variation in the invasion probability (P<0.05. Since 2001, the number of new records of the nematode was multiplied by a factor of 5 and the spread distance by a factor of 2. We combined a diffusion model to describe the short distance spread with a stochastic, individual based model to describe the long distance jumps. This combined model generated an error of only 13% when used to predict the presence of the nematode. Under two climate scenarios (stable climate or moderate warming, projections of the invasion probability suggest that this pest could expand its distribution 40-55% by 2025. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that human-induced dispersal plays a fundamental role in the spread of the pinewood nematode, and appropriate control measures should be taken to stop or slow its expansion. This model can be applied to Europe, where the nematode had been introduced later, and is currently expanding its distribution. Similar models could also be derived for other species that could be

  3. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, J.D., E-mail: jennifer.cohen@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Babiarz, J.E., E-mail: joshua.babiarz@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Abrams, R.M., E-mail: rory.abrams@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Guo, L., E-mail: liang.guo@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Kameoka, S., E-mail: sei.kameoka@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Chiao, E., E-mail: eric.chiao@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Taunton, J., E-mail: taunton@cmp.ucsf.edu [Howard Hughes Medical Institute, Cellular and Molecular Pharmacology, University California San Francisco, San Francisco, CA 94158 (United States); Kolaja, K.L., E-mail: kyle.kolaja@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States)

    2011-11-15

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC{sub 50} in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 {mu}M concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 {mu}M. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: Black-Right-Pointing-Pointer Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes Black

  4. Modulation of interleukin-1beta mediated inflammatory response in human astrocytes by flavonoids: implications in neuroprotection.

    Science.gov (United States)

    Sharma, Vivek; Mishra, Mamata; Ghosh, Soumya; Tewari, Richa; Basu, Anirban; Seth, Pankaj; Sen, Ellora

    2007-06-15

    The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neuronal death in CNS injuries and neurodegenerative pathologies, and astrocytes have been implicated as the primary mediators of IL-1beta induced neuronal death. As astrocytes play an important role in supporting the survival and functions of neurons, we investigated the effect of plant flavonoids quercetin and luteolin, with known anti-inflammatory properties in modulating the response of human astrocytes to IL-1beta for therapeutic intervention. Flavonoids significantly decreased the release of reactive oxygen species (ROS) from astrocytes stimulated with IL-1beta. This decrease was accompanied by an increase in expression of superoxide dismutase (SOD-1) and thioredoxin (TRX1)-mediators associated with protection against oxidative stress. Flavonoids not only modulated the expression of astrocytes specific molecules such as glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and ceruloplasmin (CP) both in the presence and absence of IL-1beta but also decreased the elevated levels of proinflammatory cytokine interleukin-6 (IL-6) and chemokines interleukin-8 (IL-8), interferon-inducible protein (IP-10), monocyte-chemoattractant protein-1 (MCP-1), and RANTES from IL-1beta activated astrocytes. Significant decrease in neuronal apoptosis was observed in neurons cultured in conditioned medium obtained from astrocytes treated with a combination of IL-1beta and flavonoids as compared to that treated with IL-1beta alone. Our result suggests that by (i) enhancing the potential of activated astrocytes to detoxify free radical, (ii) reducing the expression of proinflammatory cytokines and chemokines, and (iii) modulating expression of mediators associated with enhanced physiological activity of astrocyte in response to injury, flavonoids confer (iv) protection against IL-1beta induced astrocyte mediated neuronal damage.

  5. EP2 receptor mediates PGE2-induced cystogenesis of human renal epithelial cells.

    Science.gov (United States)

    Elberg, Gerard; Elberg, Dorit; Lewis, Teresa V; Guruswamy, Suresh; Chen, Lijuan; Logan, Charlotte J; Chan, Michael D; Turman, Martin A

    2007-11-01

    Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E2 (PGE2) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE2 directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE2 effect has not been characterized. Our goal is to define the PGE2 receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE2 induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP2 receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE2-selective analogs further demonstrates that EP2 mediates cAMP formation and cystogenesis. Functional evidence for a role of EP2 receptor in mediating cAMP signaling was also provided by inhibiting EP2 receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE2 produced in cyst fluid binds to adjacent EP2 receptors located on the apical side of cysts and stimulates EP2 receptor expression. PGE2 binding to EP2 receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP2 receptor in mediating the PGE2 effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.

  6. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription

    Science.gov (United States)

    Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M.; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T.; Wilczynski, Grzegorz M.; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

    2015-01-01

    Summary Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced ChIA-PET strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CTCF and RNAPII with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes towards CTCF-foci for coordinated transcription. Furthermore, we show that haplotype-variants and allelic-interactions have differential effects on chromosome configuration influencing gene expression and may provide mechanistic insights into functions associated with disease susceptibility. 3D-genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D-genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. PMID:26686651

  7. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

    Science.gov (United States)

    Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

    2015-12-17

    Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

  8. LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.

    Science.gov (United States)

    Luz, Simão; Cihil, Kristine M; Brautigan, David L; Amaral, Margarida D; Farinha, Carlos M; Swiatecka-Urban, Agnieszka

    2014-05-23

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.

  9. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts

    Science.gov (United States)

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-01-01

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal–placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN+CD14+CD1a− phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4+CD25+Foxp3+ Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal–fetal interface. PMID:26857012

  10. Antigen binding of human IgG Fabs mediate ERK-associated proliferation of human breast cancer cells.

    Science.gov (United States)

    Wen, Yue-Jin; Mancino, Anne; Pashov, Anastas; Whitehead, Tracy; Stanley, Joseph; Kieber-Emmons, Thomas

    2005-02-01

    Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.

  11. Induction of interleukin-6 production by ultraviolet radiation in normal human epidermal keratinocytes and in a human keratinocyte cell line is mediated by DNA damage

    NARCIS (Netherlands)

    Petit-Frère, C.; Clingen, P.H.; Grewe, M.; Krutmann, J.; Roza, L.; Arlett, C.F.; Green, M.H.L.

    1998-01-01

    The sunburn reaction is the most common consequence of human exposure to ultraviolet radiation (UVR), and is mediated at least in part by interleukin- 6 (IL-6). The aim of this study was to determine if DNA is a major chromophore involved in the induction of IL-6 following UV irradiation of a human

  12. Dexamethasone inhibits repair of human airway epithelial cells mediated by glucocorticoid-induced leucine zipper (GILZ.

    Directory of Open Access Journals (Sweden)

    Jingyue Liu

    Full Text Available BACKGROUND: Glucocorticoids (GCs are a first-line treatment for asthma for their anti-inflammatory effects, but they also hinder the repair of airway epithelial injury. The anti-inflammatory protein GC-induced leucine zipper (GILZ is reported to inhibit the activation of the mitogen-activated protein kinase (MAPK-extracellular-signal-regulated kinase (ERK signaling pathway, which promotes the repair of airway epithelial cells around the damaged areas. We investigated whether the inhibition of airway epithelial repair imposed by the GC dexamethasone (DEX is mediated by GILZ. METHODS: We tested the effect of DEX on the expressions of GILZ mRNA and GILZ protein and the MAPK-ERK signaling pathway in human airway epithelial cells, via RT-PCR and Western blot. We further evaluated the role of GILZ in mediating the effect of DEX on the MAPK-ERK signaling pathway and in airway epithelium repair by utilizing small-interfering RNAs, MTT, CFSE labeling, wound-healing and cell migration assays. RESULTS: DEX increased GILZ mRNA and GILZ protein levels in a human airway epithelial cell line. Furthermore, DEX inhibited the phosphorylation of Raf-1, Mek1/2, Erk1/2 (components of the MAPK-ERK signaling pathway, proliferation and migration. However, the inhibitory effect of DEX was mitigated in cells when the GILZ gene was silenced. CONCLUSIONS: The inhibition of epithelial injury repair by DEX is mediated in part by activation of GILZ, which suppressed activation of the MAPK-ERK signaling pathway, proliferation and migration. Our study implicates the involvement of DEX in this process, and furthers our understanding of the dual role of GCs.

  13. MPLA inhibits release of cytotoxic mediators from human neutrophils while preserving efficient bacterial killing.

    Science.gov (United States)

    Ruchaud-Sparagano, Marie-Hélène; Mills, Ross; Scott, Jonathan; Simpson, A John

    2014-10-01

    Monophosphoryl lipid A (MPLA) is a lipopolysaccharides (LPS) derivative associated with neutrophil-dependent anti-inflammatory outcomes in animal models of sepsis. Little is known about the effect of MPLA on neutrophil function. This study sought to test the hypothesis that MPLA would reduce release of cytotoxic mediators from neutrophils without impairing bacterial clearance. Neutrophils were isolated from whole blood of healthy volunteers. The effects of MPLA and LPS on autologous serum-opsonised Pseudomonas aeruginosa killing by neutrophils and phagocytosis of autologous serum-opsonised zymosan were examined. Neutrophil oxidative burst, chemotaxis, enzyme and cytokine release as well as Toll-like receptor 4 (TLR4) expression were assessed following exposure to LPS or MPLA. LPS, but not MPLA, induced significant release of superoxide and myeloperoxidase from neutrophils. However, MPLA did not impair neutrophil capacity to ingest microbial particles and kill P. aeruginosa efficiently. MPLA was directly chemotactic for neutrophils, involving TLR4, p38 mitogen-activated protein kinase and tyrosine and alkaline phosphatases. LPS, but not MPLA, impaired N-formyl-methionyl-leucyl phenylalanine-directed migration of neutrophils, increased surface expression of TLR4, increased interleukin-8 release and strongly activated the myeloid differentiation primary response 88 pathway. Phosphoinositide 3-kinase inhibition significantly augmented IL-8 release from MPLA-treated neutrophils. The addition of MPLA to LPS-preincubated neutrophils led to a significant reduction in LPS-mediated superoxide release and TLR4 surface expression. Collectively, these findings suggest that MPLA directs efficient chemotaxis and bacterial killing in human neutrophils without inducing extracellular release of cytotoxic mediators and suggest that MPLA warrants further attention as a potential therapeutic in human sepsis.

  14. BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells

    Directory of Open Access Journals (Sweden)

    Liu Jun

    2004-07-01

    Full Text Available Abstract Background BAK (Bcl-2 homologous antagonist/killer is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro. Methods Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53 and MKN-28 (mutant-type p53. RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities. Results BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G0/G1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45, or in p53 mutant-type (MKN-28 gastric cancer cells. Conclusions The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies.

  15. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-11-04

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  16. Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Wei-ying ZHANG; Fu-qing XU; Chang-liang SHAN; Rong XIANG; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model.Methods: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes, Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2).Results: The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion.Conclusion: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the car-cinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.

  17. α6 subunit-containing nicotinic receptors mediate low-dose ethanol effects on ventral tegmental area neurons and ethanol reward.

    Science.gov (United States)

    Steffensen, Scott C; Shin, Samuel I; Nelson, Ashley C; Pistorius, Stephanie S; Williams, Stephanie B; Woodward, Taylor J; Park, Hyun Jung; Friend, Lindsey; Gao, Ming; Gao, Fenfei; Taylor, Devin H; Foster Olive, M; Edwards, Jeffrey G; Sudweeks, Sterling N; Buhlman, Lori M; Michael McIntosh, J; Wu, Jie

    2017-09-13

    Dopamine (DA) neuron excitability is regulated by inhibitory GABAergic synaptic transmission and modulated by nicotinic acetylcholine receptors (nAChRs). The aim of this study was to evaluate the role of α6 subunit-containing nAChRs (α6*-nAChRs) in acute ethanol effects on ventral tegmental area (VTA) GABA and DA neurons. α6*-nAChRs were visualized on GABA terminals on VTA GABA neurons, and α6*-nAChR transcripts were expressed in most DA neurons, but only a minority of VTA GABA neurons from GAD67 GFP mice. Low concentrations of ethanol (1-10 mM) enhanced GABAA receptor (GABAA R)-mediated spontaneous and evoked inhibition with blockade by selective α6*-nAChR antagonist α-conotoxins (α-Ctxs) and lowered sensitivity in α6 knock-out (KO) mice. Ethanol suppression of VTA GABA neuron firing rate in wild-type mice in vivo was significantly reduced in α6 KO mice. Ethanol (5-100 mM) had no effect on optically evoked GABAA R-mediated inhibition of DA neurons, and ethanol enhancement of VTA DA neuron firing rate at high concentrations was not affected by α-Ctxs. Ethanol conditioned place preference was reduced in α6 KO mice compared with wild-type controls. Taken together, these studies indicate that relatively low concentrations of ethanol act through α6*-nAChRs on GABA terminals to enhance GABA release onto VTA GABA neurons, in turn to reduce GABA neuron firing, which may lead to VTA DA neuron disinhibition, suggesting a possible mechanism of action of alcohol and nicotine co-abuse. © 2017 Society for the Study of Addiction.

  18. Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin.

    Science.gov (United States)

    Abbot, Emily L; Smith, Wendy D; Siou, Gerard P S; Chiriboga, Carlos; Smith, Rebecca J; Wilson, Janet A; Hirst, Barry H; Kehoe, Michael A

    2007-07-01

    Very little is known about the biological functions of pili that have recently been found to be expressed by important Gram-positive pathogens such as Corynebacterium diphtheriae, Streptococcus agalacticae, S. pneumoniae and S. pyogenes. Using various ex vivo tissue and cellular models, here we show that pili mediate adhesion of serotype M1 S. pyogenes strain SF370 to both human tonsil epithelium and primary human keratinocytes, which represent the two main sites of infection by this human-specific pathogen. Mutants lacking minor pilus subunits retained the ability to express cell-surface pili, but these were functionally defective. In contrast to above, pili were not required for S. pyogenes adhesion to either immortalized HEp-2 or A549 cells, highlighting an important limitation of these extensively used adhesion/invasion models. Adhering bacteria were internalized very effectively by both HEp-2 and A549 cells, but not by tonsil epithelium or primary keratinocytes. While pili acted as the primary adhesin, the surface M1 protein clearly enhanced adhesion to tonsil, but surprisingly, had the opposite effect on adhesion to keratinocytes. These studies provide clear evidence that S. pyogenes pili display an adhesive specificity for clinically relevant human tissues and are likely to play a critical role in the initial stages of infection.

  19. Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

    Institute of Scientific and Technical Information of China (English)

    PAN Xin-ting; ZHU Qing-yun; LI De-chun; YANG Ji-cheng; ZHANG Zi-xiang; ZHU Xing-guo; ZHAO Hua

    2008-01-01

    Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhlL-24) on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n=10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0x1010 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P <0.05). The intratumoral MVD decreased significantly in the treated tumors (P <0.05).Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

  20. KLF5 regulates infection- and inflammation-induced pro-labour mediators in human myometrium.

    Science.gov (United States)

    Lappas, Martha

    2015-05-01

    The transcription factor Kruppel-like factor 5 (KLF5) has been shown to associate with nuclear factor kappa B (NFκB) to regulate genes involved in inflammation. However, there are no studies on the expression and regulation of KLF5 in the processes of human labour and delivery. Thus, the aims of this study were to determine the effect of i) human labour on KLF5 expression in both foetal membranes and myometrium; ii) the pro-inflammatory cytokine interleukin 1 beta (IL1β), bacterial product flagellin and the viral dsRNA analogue poly(I:C) on KLF5 expression and iii) KLF5 knockdown by siRNA in human myometrial primary cells on pro-inflammatory and pro-labour mediators. In foetal membranes, there was no effect of term or preterm labour on KLF5 expression. In myometrium, the term labour was associated with an increase in nuclear KLF5 protein expression. Moreover, KLF5 expression was also increased in myometrial cells treated with IL1β, flagellin or poly(IC), likely factors contributing to preterm birth. KLF5 silencing in myometrial cells significantly decreased IL1β-induced cytokine expression (IL6 and IL8 mRNA expression and release), COX2 mRNA expression, and subsequent release of prostaglandins PGE2 and PGF2 α. KLF5 silencing also significantly reduced flagellin- and poly(I:C)-induced IL6 and IL8 mRNA expression. Lastly, IL1β-, flagellin- and poly(I:C)-stimulated NFκB transcriptional activity was significantly suppressed in KLF5-knockout myometrial cells. In conclusion, this study describes novel data in which KLF5 is increased in labouring myometrium, and KLF5 silencing decreased inflammation- and infection-induced pro-labour mediators. © 2015 Society for Reproduction and Fertility.

  1. Specialized pro-resolving mediators (SPMs) inhibit human B-cell IgE production

    Science.gov (United States)

    Kim, Nina; Ramon, Sesquile; Thatcher, Thomas H.; Woeller, Collynn F.; Sime, Patricia J.; Phipps, Richard P.

    2015-01-01

    Specialized pro-resolving lipid mediators (SPMs) constitute a recently recognized class of bioactive molecules which promote the resolution of inflammation. We recently reported that the SPMs resolvin D1 (RvD1) and 17-hydroxydocosahexaenoic acid (17-HDHA) promote the differentiation of IgG-secreting B cells and enhance antibody-mediated immune responses. However, there is an important knowledge gap regarding whether or not SPMs regulate human B-cell IgE production, which is the key effector in diseases such as asthma and allergy. Therefore we investigated whether a panel of diverse SPMs influences B-cell IgE production. An important finding was that 17-HDHA and RvD1 inhibit IgE production by human B cells and suppress the differentiation of naïve B cells into IgE-secreting cells by specifically blocking epsilon germline transcription (εGLT). This effect is specific to human IgE, as the SPMs do not inhibit production of IgM and IgG and did not suppress other IL-4-upregulated genes. 17-HDHA and RvD1 act by stabilizing the transcriptional repressor Bcl-6, which competes with STAT6 for binding at the εGLT promoter. Overall, these new findings demonstrate that certain SPMs inhibit the differentiation of IgE-producing B cells, without being broadly immune-suppressive, representing a novel class of potential therapeutics for IgE-driven diseases such as asthma and allergy. PMID:26474728

  2. HABP2 is a novel regulator of hyaluronan-mediated human lung cancer progression

    Directory of Open Access Journals (Sweden)

    Tamara eMirzapoiazova

    2015-07-01

    Full Text Available Background: Lung cancer is a devastating disease with limited treatment options. Many lung cancers have changes in their microenvironment including upregulation of the extracellular matrix glycosaminoglycan, hyaluronan (HA, which we have previously demonstrated can regulate the activity of the extracellular serine protease, Hyaluronan Binding Protein 2 (HABP2. This study examined the functional role of HABP2 on HA-mediated human lung cancer dynamics.Methods: Immunohistochemical analysis was performed on lung cancer patient samples using anti-HABP2 antibody. Stable control, shRNA and HABP2 overexpressing human lung adenocarcinoma cells were evaluated using immunoblot analysis, migration, extravasation and urokinase plasminogen activator (uPA activation assays with or without high molecular weight HA (HMW-HA or low molecular weight HA (LMW-HA. In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs.Results: We provide evidence that HABP2 is an important regulator of lung cancer progression. HABP2 expression was increased in several subtypes of patient non-small cell lung cancer samples. Further, HABP2 overexpression increased LMW-HA-induced uPA activation, migration and extravasation in human lung adenocarcinoma cells. In vivo, overexpression of HABP2 in human lung adenocarcinoma cells increased primary tumor growth rates in nude mice by ~2 fold and lung metastasis by ~10 fold compared to vector control cells (n=5 per condition.Conclusions: Our data suggests a possible direct effect of HABP2 on uPA activation and lung cancer progression. Our observations suggest that exploration of HABP2 in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

  3. RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%~ 72.5%) than in supernatant system (33.1%~ 46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

  4. Lysophosphatidic acid mediates myeloid differentiation within the human bone marrow microenvironment.

    Directory of Open Access Journals (Sweden)

    Denis Evseenko

    Full Text Available Lysophosphatidic acid (LPA is a pleiotropic phospholipid present in the blood and certain tissues at high concentrations; its diverse effects are mediated through differential, tissue specific expression of LPA receptors. Our goal was to determine if LPA exerts lineage-specific effects during normal human hematopoiesis. In vitro stimulation of CD34+ human hematopoietic progenitors by LPA induced myeloid differentiation but had no effect on lymphoid differentiation. LPA receptors were expressed at significantly higher levels on Common Myeloid Progenitors (CMP than either multipotent Hematopoietic Stem/Progenitor Cells (HSPC or Common Lymphoid Progenitors (CLP suggesting that LPA acts on committed myeloid progenitors. Functional studies demonstrated that LPA enhanced migration, induced cell proliferation and reduced apoptosis of isolated CMP, but had no effect on either HSPC or CLP. Analysis of adult and fetal human bone marrow sections showed that PPAP2A, (the enzyme which degrades LPA was highly expressed in the osteoblastic niche but not in the perivascular regions, whereas Autotaxin (the enzyme that synthesizes LPA was expressed in perivascular regions of the marrow. We propose that a gradient of LPA with the highest levels in peri-sinusoidal regions and lowest near the endosteal zone, regulates the localization, proliferation and differentiation of myeloid progenitors within the bone marrow marrow.

  5. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

    Science.gov (United States)

    Song, Erwei; Lee, Sang-Kyung; Dykxhoorn, Derek M; Novina, Carl; Zhang, Dong; Crawford, Keith; Cerny, Jan; Sharp, Phillip A; Lieberman, Judy; Manjunath, N; Shankar, Premlata

    2003-07-01

    Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

  6. Observation and prediction of recurrent human translocations mediated by NAHR between nonhomologous chromosomes.

    Science.gov (United States)

    Ou, Zhishuo; Stankiewicz, Paweł; Xia, Zhilian; Breman, Amy M; Dawson, Brian; Wiszniewska, Joanna; Szafranski, Przemyslaw; Cooper, M Lance; Rao, Mitchell; Shao, Lina; South, Sarah T; Coleman, Karlene; Fernhoff, Paul M; Deray, Marcel J; Rosengren, Sally; Roeder, Elizabeth R; Enciso, Victoria B; Chinault, A Craig; Patel, Ankita; Kang, Sung-Hae L; Shaw, Chad A; Lupski, James R; Cheung, Sau W

    2011-01-01

    Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were analyzed. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ∼359-kb and ∼215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24 bp within interchromosomal paralogous LCRs of ∼130 kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation formation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 1143 interchromosomal LCR substrate pairs, >5 kb in size and sharing >94% sequence identity that can potentially mediate chromosomal translocations. Additional evidence for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55 bp in ∼7.8-kb paralogous subunits of 95.3% sequence identity located in the ∼579-kb (chr 8) and ∼287-kb (chr 12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations t(4;11) and t(8;12) and potentially many other interchromosomal translocations throughout the human genome. Furthermore, we provide a computationally determined genome-wide "recurrent translocation map."

  7. Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation.

    Science.gov (United States)

    Rius, Maria; Stresemann, Carlo; Keller, Daniela; Brom, Manuela; Schirrmacher, Esther; Keppler, Dietrich; Lyko, Frank

    2009-01-01

    The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.

  8. STAT6 Mediates Interleukin-4 Growth Inhibition in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Jennifer L. Gooch

    2002-01-01

    Full Text Available In addition to acting as a hematopoietic growth factor, interleukin-4 (IL-4 inhibits growth of some transformed cells in vitro and in vivo. In this study, we show that insulin receptor substrate (IRS-1, IRS-2, and signal transducer and activator of transcription 6 (STAT6 are phosphorylated following IL-4 treatment in MCF-7 breast cancer cells. STAT6 DNA binding is enhanced by IL-4 treatment. STAT6 activation occurs even after IRS-1 depletion, suggesting the two pathways are independent. To examine the role of STAT6 in IL-4-mediated growth inhibition and apoptosis, a fulllength STAT6 cDNA was transfected into MCF-7 cells. Transient overexpression of STAT6 resulted in both cytoplasmic and nuclear expression of the protein, increased DNA binding in response to IL-4, and increased transactivation of an IL-4 responsive promoter. In STAT6-transfected cells, basal proliferation was reduced whereas apoptosis was increased. Finally, stable expression of STAT6 resulted in reduced foci formation compared to vector-transfected cells alone. These results suggest STAT6 is required for IL-4mediated growth inhibition and induction of apoptosis in human breast cancer cells.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  10. Fibronectin-integrin mediated signaling in human cervical cancer cells (SiHa).

    Science.gov (United States)

    Maity, Gargi; Fahreen, Shabana; Banerji, Aniruddha; Roy Choudhury, Paromita; Sen, Triparna; Dutta, Anindita; Chatterjee, Amitava

    2010-03-01

    Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

  11. E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

    Science.gov (United States)

    Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

    2016-11-01

    Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

  12. Trefoil factor 3 is oncogenic and mediates anti-estrogen resistance in human mammary carcinoma.

    Science.gov (United States)

    Kannan, Nagarajan; Kang, Jian; Kong, Xiangjun; Tang, Jianzhong; Perry, Jo K; Mohankumar, Kumarasamypet M; Miller, Lance D; Liu, Edison T; Mertani, Hichem C; Zhu, Tao; Grandison, Prudence M; Liu, Dong-Xu; Lobie, Peter E

    2010-12-01

    We report herein that trefoil factor 3 (TFF3) is oncogenic and mediates anti-estrogen resistance in human mammary carcinoma. Forced expression of TFF3 in mammary carcinoma cells increased cell proliferation and survival, enhanced anchorage-independent growth, and promoted migration and invasion. Moreover, forced expression of TFF3 increased tumor size in xenograft models. Conversely, depletion of endogenous TFF3 with small interfering RNA (siRNA) decreased the oncogenicity and invasiveness of mammary carcinoma cells. Neutralization of secreted TFF3 by antibody promoted apoptosis, decreased cell growth in vitro, and arrested mammary carcinoma xenograft growth. TFF3 expression was significantly correlated to decreased survival of estrogen receptor (ER)-positive breast cancer patients treated with tamoxifen. Forced expression of TFF3 in mammary carcinoma cells increased ER transcriptional activity, promoted estrogen-independent growth, and produced resistance to tamoxifen and fulvestrant in vitro and to tamoxifen in xenograft models. siRNA-mediated depletion or antibody inhibition of TFF3 significantly enhanced the efficacy of antiestrogens. Increased TFF3 expression was observed in tamoxifen-resistant (TAMR) cells and antibody inhibition of TFF3 in TAMR cells improved tamoxifen sensitivity. Functional antagonism of TFF3 therefore warrants consideration as a novel therapeutic strategy for mammary carcinoma.

  13. Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

    Science.gov (United States)

    Sheibani, Sara; Jones, Natalie K; Eid, Rawan; Gharib, Nada; Arab, Nagla T T; Titorenko, Vladimir; Vali, Hojatollah; Young, Paul A; Greenwood, Michael T

    2015-08-01

    We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. The human desmin promoter drives robust gene expression for skeletal muscle stem cell-mediated gene therapy.

    Science.gov (United States)

    Jonuschies, Jacqueline; Antoniou, Michael; Waddington, Simon; Boldrin, Luisa; Muntoni, Francesco; Thrasher, Adrian; Morgan, Jennifer

    2014-01-01

    Lentiviral vectors (LVs) represent suitable candidates to mediate gene therapy for muscular dystrophies as they infect dividing and non-dividing cells and integrate their genetic material into the host genome, thereby theoretically mediating longterm expression. We evaluated the ability of LVs where a GFP reporter gene was under the control of five different promoters, to transduce and mediate expression in myogenic and non-myogenic cells in vitro and in skeletal muscle fibres and stem (satellite) cells in vivo. We further analysed lentivirally-transduced satellite cell-derived myoblasts following their transplantation into dystrophic, immunodeficient mouse muscles. The spleen focus-forming virus promoter mediated the highest gene expression in all cell types; the CBX3-HNRPA2B1 ubiquitously-acting chromatin opening element (UCOE) promoter was also active in all cells, whereas the human desmin promoter in isolation or fused with UCOE had lower activity in non-muscle cells. Surprisingly, the human skeletal muscle actin promoter was also active in immune cells. The human desmin promoter mediated robust, persistent reporter gene expression in myogenic cells in vitro, and satellite cells and muscle fibres in vivo. The human desmin promoter combined with UCOE did not significantly increase transgene expression. Therefore, our data indicate that the desmin promoter is suitable for the development of therapeutic purposes.

  15. IL-23 and T(H)17-mediated inflammation in human allergic contact dermatitis

    DEFF Research Database (Denmark)

    Larsen, Jeppe Madura; Bonefeld, Charlotte Menné; Poulsen, Steen Seier

    2009-01-01

    . OBJECTIVE: To investigate T(H)17-mediated inflammation in human beings with allergic contact dermatitis; in particular, the innate response of keratinocytes to contact allergen, the induction of allergen-specific T(H)17 cells, and the presence of T(H)17-related effector cells in inflamed skin. METHODS......-term ex vivo assay. Nickel patch test lesions and normal skin were analyzed for the expression of T(H)17-related cells and molecules by using immunohistochemistry. RESULTS: Keratinocytes were found to produce IL-23, but no detectable IL-12, in a response to nickel stimulation. Memory T cells isolated from...... peripheral blood of individuals with nickel allergy, but not healthy controls, contained T(H)17 and T(H)1 cells proliferating in response to nickel-pulsed DCs. Inflamed skin of nickel-challenged allergic individuals contained infiltrating neutrophils and cells expressing IL-17, IL-22, CCR6, and IL-22R...

  16. Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

    Science.gov (United States)

    Morozumi, Yuichi; Ino, Ryohei; Takaku, Motoki; Hosokawa, Mihoko; Chuma, Shinichiro; Kurumizaka, Hitoshi

    2012-01-01

    PSF is considered to have multiple functions in RNA processing, transcription and DNA repair by mitotic recombination. In the present study, we found that PSF is produced in spermatogonia, spermatocytes and spermatids, suggesting that PSF may also function in meiotic recombination. We tested the effect of PSF on homologous pairing by the meiosis-specific recombinase DMC1, and found that human PSF robustly stimulated it. PSF synergistically enhanced the formation of a synaptic complex containing DMC1, ssDNA and dsDNA during homologous pairing. The PSF-mediated DMC1 stimulation may be promoted by its DNA aggregation activity, which increases the local concentrations of ssDNA and dsDNA for homologous pairing by DMC1. These results suggested that PSF may function as an activator for the meiosis-specific recombinase DMC1 in higher eukaryotes. PMID:22156371

  17. Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia.

    Science.gov (United States)

    Lau, Yee-Ling; Lai, Meng-Yee; Fong, Mun-Yik; Jelip, Jenarun; Mahmud, Rohela

    2016-02-01

    The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent.

  18. Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma.

    Science.gov (United States)

    Verstraete, Kenneth; Peelman, Frank; Braun, Harald; Lopez, Juan; Van Rompaey, Dries; Dansercoer, Ann; Vandenberghe, Isabel; Pauwels, Kris; Tavernier, Jan; Lambrecht, Bart N; Hammad, Hamida; De Winter, Hans; Beyaert, Rudi; Lippens, Guy; Savvides, Savvas N

    2017-04-03

    The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.

  19. Effects of large conductance Ca(2+)-activated K(+) channels on nitroglycerin-mediated vasorelaxation in humans

    DEFF Research Database (Denmark)

    Gruhn, Nicolai; Boesgaard, Søren; Eiberg, Jonas

    2002-01-01

    Nitric oxide (NO)-induced vasorelaxation and the regulation of endothelial superoxide anion levels is partly mediated by vascular large conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Nitroglycerin acts through the release of NO and its effect is modulated by changes in endothelial superoxide...... levels. This study examines the effect of BK(Ca) channel blockade on nitroglycerin-induced vasorelaxation in human arterial and venous vascular segments and whether responses to BK(Ca) channel blockade are influenced by the development of venous nitroglycerin tolerance. Dose-relaxation curves...... suggest that primarily arterial effects of nitroglycerin are significantly inhibited by changes in the activity of the endothelial BK(Ca) channels. Although endothelial BK(Ca) are likely regulators of mechanisms underlying arterial tolerance development to nitroglycerin, they do not appear to play a role...

  20. Conjugation of gold nanoparticles and recombinant human endostatin modulates vascular normalization via interruption of anterior gradient 2-mediated angiogenesis.

    Science.gov (United States)

    Pan, Fan; Yang, Wende; Li, Wei; Yang, Xiao-Yan; Liu, Shuhao; Li, Xin; Zhao, Xiaoxu; Ding, Hui; Qin, Li; Pan, Yunlong

    2017-07-01

    Several studies have revealed the potential of normalizing tumor vessels in anti-angiogenic treatment. Recombinant human endostatin is an anti-angiogenic agent which has been applied in clinical tumor treatment. Our previous research indicated that gold nanoparticles could be a nanoparticle carrier for recombinant human endostatin delivery. The recombinant human endostatin-gold nanoparticle conjugates normalized vessels, which improved chemotherapy. However, the mechanism of recombinant human endostatin-gold nanoparticle-induced vascular normalization has not been explored. Anterior gradient 2 has been reported to be over-expressed in many malignant tumors and involved in tumor angiogenesis. To date, the precise efficacy of recombinant human endostatin-gold nanoparticles on anterior gradient 2-mediated angiogenesis or anterior gradient 2-related signaling cohort remained unknown. In this study, we aimed to explore whether recombinant human endostatin-gold nanoparticles could normalize vessels in metastatic colorectal cancer xenografts, and we further elucidated whether recombinant human endostatin-gold nanoparticles could interrupt anterior gradient 2-induced angiogenesis. In vivo, it was indicated that recombinant human endostatin-gold nanoparticles increased pericyte expression while inhibit vascular endothelial growth factor receptor 2 and anterior gradient 2 expression in metastatic colorectal cancer xenografts. In vitro, we uncovered that recombinant human endostatin-gold nanoparticles reduced cell migration and tube formation induced by anterior gradient 2 in human umbilical vein endothelial cells. Treatment with recombinant human endostatin-gold nanoparticles attenuated anterior gradient 2-mediated activation of MMP2, cMyc, VE-cadherin, phosphorylation of p38, and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human umbilical vein endothelial cells. Our findings demonstrated recombinant human endostatin-gold nanoparticles might normalize

  1. CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

    Science.gov (United States)

    Kang, Yu; Zheng, Bo; Shen, Bin; Chen, Yongchang; Wang, Lei; Wang, Jianying; Niu, Yuyu; Cui, Yiqiang; Zhou, Jiankui; Wang, Hong; Guo, Xuejiang; Hu, Bian; Zhou, Qi; Sha, Jiahao; Ji, Weizhi; Huang, Xingxu

    2015-12-20

    Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/β-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting.

  2. Aminolevulinic Acid-Mediated Photodynamic Therapy Causes Cell Death in MG-63 Human Osteosarcoma Cells.

    Science.gov (United States)

    White, Bradley; Rossi, Vince; Baugher, Paige J

    2016-09-01

    The aim of this study was to test the efficacy of aminolevulinic acid-mediated photodynamic therapy (PDT) against the human osteosarcoma cell line MG-63. Osteosarcoma is the most common type of primary malignant bone tumor diagnosed in the United States among adolescents and children. Treatments for osteosarcoma often result in diminished limb use or amputation. Because ALA-mediated PDT exhibits dual specificity in the context of tumor killing, this therapy could represent a less invasive, but effective, treatment for this disease. To assess ALA dark toxicity in MG-63 cells, cells were incubated with varying concentrations of ALA, and cell viability was determined by crystal violet assay. Protoporphyrin IX (PpIX) accumulation was assessed subsequent to ALA incubation at various concentrations using spectrofluorometry. Cell death subsequent to ALA-PDT was determined by illuminating cells at a wavelength of 635 nm at various light intensities subsequent to ALA incubation. Cell viability was assessed using the MTT assay. ALA dark toxicity was observed only at the highest concentrations of 2, 5, and 10 mM. Maximal PpIX concentration was observed at 0.5 and 1 mM ALA, subsequent to a 24-h incubation. Maximal cell death with minimal light toxicity was observed at 0.5 and 1 mM ALA after illumination with 0.6 and 3 J/cm(2) light. Collectively, our data indicate that ALA-PDT can result in the death of MG-64 human osteosarcoma cells in vitro.

  3. Aquaporin-1-Mediated Effects of Low Level He-Ne Laser Irradiation on Human Erythrocytes

    Directory of Open Access Journals (Sweden)

    Gang-Yue Luo

    2012-01-01

    Full Text Available The role of membrane aquaporin-1 (APQ-1 in the photobiomodulation (PBM on erythrocyte deformability will be studied in this paper with human dehydrated erythrocytes as echinocytic shape alterations lead to decreased cellular deformability. Human dehydrated erythrocytes were irradiated with low intensity He-Ne laser irradiation (LHNL at 0.9, 1.8, 2.7, and 4.4 mW/cm2 for 5, 15, and 30 min, respectively, and APQ-1 inhibitor, 0.2 μmol/L HgCl2, was used to study the role of APQ-1 in mediating PBM with LHNL at 4.4 mW/cm2 for 5 min. Comprehensive morphological parameters of an intact cell such as contact area, perimeter, roundness and erythrocyte elongation index (EEI were measured to characterize erythrocyte deformability with fast micro multi-channel spectrophotometer. It was observed that the dosage of LHNL improvement of the morphological parameters of dehydrated erythrocytes was morphological-parameter-dependent, but the Bunsen-Roscoe rule did not hold for roundness. The LHNL at 4.4 mW/cm2 for 5 min significantly improved the contact area (P<0.05 and EEI (P<0.05 of the dehydrated erythrocytes, but the improvement was significantly inhibited by 0.2 μmol/L HgCl2 (P<0.05. It was concluded that AQP-1 might mediate the effects of LHNL on erythrocyte deformability, which supports the membranotropic mechanism of PBM.

  4. Biodiesel exhaust-induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells.

    Science.gov (United States)

    Mullins, Benjamin J; Kicic, Anthony; Ling, Kak-Ming; Mead-Hunter, Ryan; Larcombe, Alexander N

    2016-01-01

    Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow-sulfur-diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)-6, IL-8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL-8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size.

  5. Crop domestication, global human-mediated migration, and the unresolved role of geography in pest control

    Directory of Open Access Journals (Sweden)

    Yolanda H. Chen

    2016-05-01

    Full Text Available Abstract Ecological pest management seeks to improve pest control through the manipulation of ecological processes that promote natural enemies and suppress pests. These approaches can involve cultural practices such as reduced tillage, increased use of non-crop plants that provide food and shelter for natural enemies, and intercropping to enhance the abundance and diversity of natural enemies. A major assumption of ecological pest management is that these activities can be equally effective for all insect herbivores. Here, I propose that these strategies may only be effective for a subset of pests and geographic regions because most insect pests have complex evolutionary histories that make them difficult to manage. I discuss how crop domestication and human-mediated migration are major evolutionary events that shape the geography of interactions between plants, herbivores, and natural enemies. Insect herbivores can evolve to be pests through three major modes: 1 herbivores associated with the crop wild ancestor may shift onto the domesticated crop, 2 herbivores may host-shift from native host plants onto an introduced crop, or 3 human-mediated migration can introduce insect pests into new cropping regions. The resulting geographic structure can influence the success of pest management by altering ecological factors such as: species distributions, patterns of biodiversity, community structure, and natural enemy attack rates. I discuss how the different modes of insect pest evolution structure a set of relevant questions and approaches for ecological pest management. By acknowledging how agricultural history and geography shape the ecology and evolution of insect pests, we may collectively develop a better capacity to identify where and how ecological pest management approaches can be most broadly effective.

  6. Substrate specificity of human ABCC4 (MRP4)-mediated cotransport of bile acids and reduced glutathione.

    Science.gov (United States)

    Rius, Maria; Hummel-Eisenbeiss, Johanna; Hofmann, Alan F; Keppler, Dietrich

    2006-04-01

    The multidrug resistance protein ABCC4 (MRP4), a member of the ATP-binding cassette superfamily, mediates ATP-dependent unidirectional efflux of organic anions out of cells. Previous studies showed that human ABCC4 is localized to the sinusoidal membrane of hepatocytes and mediates, among other substrates, the cotransport of reduced glutathione (GSH) with bile acids. In the present study, using inside-out membrane vesicles, we demonstrated that human ABCC4 in the presence of physiological concentrations of GSH has a high affinity for the taurine and glycine conjugates of the common natural bile acids as well as the unconjugated bile acid cholate. Chenodeoxycholyltaurine and chenodeoxycholylglycine were the GSH cosubstrates with the highest affinities for ABCC4, with K(m) values of 3.6 and 5.9 microM, respectively. Ursodeoxycholyltaurine and ursodeoxycholylglycine were cotransported together with GSH by ABCC4 with K(m) values of 7.8 and 12.5 microM, respectively, but no transport of ursodeoxycholate and deoxycholate was observed. The simultaneous transport of labeled GSH and cholyltaurine or cholylglycine was demonstrated in double-labeled cotransport experiments with a bile acid-to-GSH ratio of approximately 1:22. K(m) values of the bile acids for ABCC4 were in a range similar to those reported for the canalicular bile salt export pump ABCB11. Under physiological conditions, the sinusoidal ABCC4 may compete with canalicular ABCB11 for bile acids and thereby play a key role in determining the hepatocyte concentration of bile acids. In cholestatic conditions, ABCC4 may become a key pathway for efflux of bile acids from hepatocytes into blood.

  7. A novel human immunoglobulin Fcγ–Fcε bifunctional fusion protein inhibits FcεRI-mediated degranulation

    OpenAIRE

    Zhu, Daocheng; Kepley, Christopher L.; Zhang, Min; Zhang, Ke; Saxon, Andrew

    2002-01-01

    Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fcε receptor 1 (FcεRI), have key roles in allergic diseases. FcεRI cross-linking stimulates the release of allergic mediators1. Mast cells and basophils co-express FcγRIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with FcεRI can block FcεRI-mediated reactivity2–4. Here we designed, expressed and tested the human basophil and mast-c...

  8. Benzyl isothiocyanate causes FoxO1-mediated autophagic death in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Dong Xiao

    Full Text Available Benzyl isothiocyanate (BITC, a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04 and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy and acidic vesicular organelles (acridine orange staining, cleavage of microtubule-associated protein 1 light chain 3 (LC3, and/or suppression of p62 (p62/SQSTM1 or sequestosome 1 expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1 in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.

  9. Muscarinic acetylcholine receptor-mediated effects in slices from human epileptogenic cortex.

    Science.gov (United States)

    Gigout, S; Wierschke, S; Lehmann, T-N; Horn, P; Dehnicke, C; Deisz, R A

    2012-10-25

    Acetylcholine has been implicated in higher cortical functions such as learning, memory and cognition, yet the cellular effects of muscarinic acetylcholine receptor (mAChR) activation are poorly understood in the human cortex. Here we investigated the effect of the mAChR agonist carbachol (CCh) and various mAChR antagonists in human cortical slices (from tissue removed during neurosurgical treatment of epilepsy) by intracellular and extracellular recordings. CCh increased neuronal firing, which was antagonised by atropine (non-selective mAChR antagonist) and pirenzepine (M(1)/M(4) mAChRs antagonist) when applied before or after CCh application. AF-DX 116 (M(2)/M(4) mAChRs antagonist) had no effect on CCh-induced increase of firing. CCh also reduced evoked excitatory postsynaptic potentials (EPSP), and the CCh-induced depression of EPSP was fully reversed by atropine. Pirenzepine reversed the depression of CCh on EPSP, but failed to prevent the depression when applied before CCh. AF-DX 116 prevented the CCh-induced depression of evoked EPSP when applied before CCh. CCh also depressed GABAergic transmission and this effect was antagonised by AF-DX 116. Xanomeline (M(1)/M(4) mAChR agonist) increased neuronal firing and decreased EPSP, but had no effect on GABAergic transmission. Reduction (with linopirdine) and enhancement (with retigabine) of the M-current (mediated by K(V)7 channels), increased and decreased neuronal firing, respectively, but had marginal effects on the evoked EPSP. Our results indicate that three pharmacologically distinct mAChRs modulate neuronal firing, glutamatergic and GABAergic transmissions in the human epileptogenic neocortex. The data are discussed towards possible implications of altered mAChR signalling in hyperexcitability and cognitive functions in the human neocortex. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide-sperm-mediated gene transfer.

    Science.gov (United States)

    Li, Lan; Shen, Wei; Min, Lingjiang; Dong, Huansheng; Sun, Yujiang; Pan, Qingjie

    2006-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins. However, it is difficult to validate whether these transgenic domestic animals are able to express the recombinant protein efficiently in their mammary glands before the birth of transgenic offspring. In the present study, a simple and efficient method was established to evaluate the functionality of animal mammary gland tissue-expressed cassettes. The gene transfer vector pGBC2LF was constructed, and the expression of human lactoferrin (LF) gene was controlled by the goat beta-casein gene 5' flanking sequence. To obtain the most efficient transfection, the influence of DNA concentration, dimethylsulfoxide (DMSO) concentration, and the ratio of linear-to-circular DNA required for associating DNA with spermatozoa were evaluated. Transfection of exogenous DNA into rabbit spermatozoa was found to be efficient using 30 microg mL(-1) DNA, DMSO at a final concentration of 3%, and a 3 : 1 ratio of linear-to-circular DNA, with 29 of 85 (34.1%) in vitro-fertilised embryos being transgenic. Using DMSO-sperm-mediated gene transfer (DMSO-SMGT), 89 rabbit offspring were produced, with 46 of these (57.1%) being transgenic. As mammary gland bioreactor models, 17 of 21 (81%) transgenic female rabbits could express human LF protein in their glands. During lactation of the transgenic rabbits, the highest level of human LF protein expressed was 153 +/- 31 microg mL(-1), and the mean expression level in all of the transgenic rabbits was 103 +/- 20 microg mL(-1) in the third week, declining gradually after this time. Our results demonstrate that transgenic rabbits produced by DMSO-SMGT were able to express human LF protein in the correct tissue.

  11. Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

    Science.gov (United States)

    Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

    2008-06-01

    Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

  12. Low CCR7-mediated migration of human monocyte derived dendritic cells in response to human respiratory syncytial virus and human metapneumovirus.

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    Cyril Le Nouën

    2011-06-01

    Full Text Available Human respiratory syncytial virus (HRSV and, to a lesser extent, human metapneumovirus (HMPV and human parainfluenza virus type 3 (HPIV3, can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV-stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to

  13. A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation.

    Science.gov (United States)

    Zhu, Daocheng; Kepley, Christopher L; Zhang, Min; Zhang, Ke; Saxon, Andrew

    2002-05-01

    Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.

  14. Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Ton, Hoai T; Smart, Amanda E; Aguilar, Brittany L; Olson, Thao T; Kellar, Kenneth J; Ahern, Gerard P

    2015-08-01

    The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.

  15. The influence of the nitrogen substitution in three cytisine derivatives as ligands for the neuronal nAChRs: a structural and theoretical study.

    Science.gov (United States)

    Bombieri, Gabriella; Meneghetti, Fiorella; Artali, Roberto; Tasso, Bruno; Canu Boido, Caterina; Sparatore, Fabio

    2008-09-01

    Three cytisine derivatives, (-)-(7R,9S)-1-phenyl-3-(cytisin-12-yl)propan-1-one (1), (-)-(7R,9S)-1-phenyl-2-(cytisin-12-yl)ethane (2), and (-)-(7R,9S)-1,2-bis(cytisin-12-yl)ethane (3), with different electronic and steric features have been characterized by X-ray analysis and theoretical calculations in order to evaluate how structural modulations affect the intrinsic binding affinity at the neuronal nicotinic receptors (nAChRs). The crystal structures of 1 and 2, which display comparable affinities, are characterized by the same conformation of the cytisine moiety with different orientations of the substituent at N2. In 3, two independent molecules have the pyridinone rings diversely oriented. This compound has a lower affinity with respect to 1 and 2, but it increases the expression of neuronal nAChRs. Compounds 1, 2, and 3 retain the key prerequisite of the classical pharmacophoric models, with sp(3)-N-atom--HBA distances close to the expected value, both in solid state and in solution (theoretical calculations), where, in contrast with the extended in the crystal state, a curled-up conformation has been found, though maintaining the N-substituent in equatorial position.

  16. Synthesis and biological evaluation of novel hybrids of highly potent and selective α4β2-Nicotinic acetylcholine receptor (nAChR) partial agonists.

    Science.gov (United States)

    Zhang, Han-Kun; Eaton, J Brek; Fedolak, Allison; Gunosewoyo, Hendra; Onajole, Oluseye K; Brunner, Dani; Lukas, Ronald J; Yu, Li-Fang; Kozikowski, Alan P

    2016-11-29

    We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile building blocks for creating potent α4β2 nicotinic acetylcholine receptor (nAChR) partial agonists with excellent selectivity over the α3β4 subtype. In our continued efforts to develop therapeutic nicotinic ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [(3)H]epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]heptane led to highly potent and selective α4β2* nAChR partial agonists with Ki values of 0.5-51.4 nM for α4β2 and negligible affinities for α3β4 and α7. Moreover, compounds 21, 25, and 30 maintained the functional profiles (EC50 and IC50 values of 15-50 nM) of the parent azetidine-containing compounds 3 and 4 in the (86)Rb(+) ion flux assays. In vivo efficacy of the most promising compound 21 was confirmed in the mouse SmartCube(®) platform and classical forced swim tests, supporting the potential use of α4β2 partial agonists for treatment of depression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Kinetics of desensitization and recovery from desensitization for human a4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells

    Institute of Scientific and Technical Information of China (English)

    Kewei D YU; Qiang LIU; Jie WU; Ronald J LUKAS

    2009-01-01

    Aim: Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human a4p2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line. Methods: Whole-cell patch clamp recordings were performed to evaluate a4p2-nAChR currents.Results: Application of 0.1 nmol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of ~70 and ~700 ms, for the onset of a4(32-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of a4p2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs'1' from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human a4p2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings'21 using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse.Conclusion: These findings indicate the existence of a4p2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.

  18. AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

    Science.gov (United States)

    Raghavan, Cibin T; Nagaraj, Ram H

    2016-08-01

    Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis.

  19. Pressure promotes angiotensin II--mediated migration of human coronary smooth muscle cells through increase in oxidative stress.

    Science.gov (United States)

    Yasunari, Kenichi; Maeda, Kensaku; Nakamura, Munehiro; Yoshikawa, Junichi

    2002-02-01

    Angiotensin II--mediated oxidative stress may play a role in the pathogenesis of coronary atherosclerosis. We examined the effects of pressure on the angiotensin II--mediated increase in oxidative stress and migration of cultured human coronary smooth muscle cells (SMCs). Increased pressure (100 mm Hg) by helium gas for 48 hours increased angiotensin II--mediated oxidative stress as evaluated by flow cytometry and SMC migration (from 15.9 +/- 2.2 to 32.0 +/- 2.4 cells per 4 high-power fields, P<0.05; n=8). The pressure-induced increases in oxidative stress observed appear to involve phospholipase D (PLD) and protein kinase C (PKC), inasmuch as the indirect PLD inhibitor suramin, at 100 micromol/L, and the PKC inhibitor chelerythrine, at 1 micromol/L, completely blocked the increase in angiotensin II--mediated oxidative stress induced by pressure. Pressure-induced increase in angiotensin II--mediated oxidative stress was inhibited by diphenylene iodonium chloride, an NADPH oxidase inhibitor, by 79% (P<0.05, n=8). Losartan (1 micromol/L), its active metabolite E3174 (1 micromol/L), and the antioxidant N-acetylcysteine (100 mmol/L) but not PD123319 (1 micromol/L) also blocked pressure-induced increases in angiotensin II--mediated oxidative stress and SMC migration (P<0.05, n=8). These findings suggest a novel cellular mechanism whereby pressure regulates the angiotensin II--mediated migration of SMCs, possibly via angiotensin II type 1 receptors, and which involves PLD-mediated, PKC-mediated, and NADPH oxidase--mediated increases in oxidative stress.

  20. Organic Anion Transporter 4-Mediated Transport of Olmesartan at Basal Plasma Membrane of Human Placental Barrier.

    Science.gov (United States)

    Noguchi, Saki; Nishimura, Tomohiro; Fujibayashi, Ayasa; Maruyama, Tetsuo; Tomi, Masatoshi; Nakashima, Emi

    2015-09-01

    Mechanisms regulating fetal transfer of olmesartan, an angiotensin-II receptor type 1 antagonist, are important as potential determinants of life-threatening adverse fetal effects. The purpose of this study was to examine the olmesartan transport mechanism through the basal plasma membrane (BM) of human syncytiotrophoblasts forming the placental barrier. Uptake of olmesartan by human placental BM vesicles was potently inhibited by dehydroepiandrosterone sulfate (DHEAS), estrone 3-sulfate, and bromosulfophthalein, which are all typical substrates of organic anion transporter (OAT) 4 localized at the BM of syncytiotrophoblasts, and was increased in the absence of chloride. In tetracycline-inducible OAT4-expressing cells, [(3) H]olmesartan uptake was increased by tetracycline treatment. Olmesartan uptake via OAT4 was concentration dependent with a Km of 20 μM, and was increased in the absence of chloride. [(3) H]Olmesartan efflux via OAT4 was also observed and was trans-stimulated by extracellular chloride and DHEAS. Thus, OAT4 mediates bidirectional transport of olmesartan and appears to regulate fetal transfer of olmesartan at the BM of syncytiotrophoblasts. Efflux transport of olmesartan via OAT4 from syncytiotrophoblasts to the fetal circulation might be facilitated in the presence of an inwardly directed physiological chloride gradient and extracellular DHEAS. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  1. O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    Energy Technology Data Exchange (ETDEWEB)

    Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, I-00146 Roma (Italy); National Institute for Infectious Diseases I.R.C.C.S. ' Lazzaro Spallanzani' , Via Portuense 292, I-00149 Roma (Italy); Gullotta, Francesca; Gioia, Magda; Coletta, Massimo [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , Via Montpellier 1, I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Piazza Umberto I 1, I-87100 Bari (Italy); Fasano, Mauro [Department of Structural and Functional Biology, and Center of Neuroscience, University of Insubria, Via Alberto da Giussano 12a, I-21052 Busto Arsizio, VA (Italy)

    2011-03-04

    Research highlights: {yields} Human serum heme-albumin displays globin-like properties. {yields} O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin. {yields} Allosteric modulation of human serum heme-albumin reactivity. {yields} Rifampicin is an allosteric effector of human serum heme-albumin. {yields} Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O{sub 2}-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10{sup -5} and 8.3 x 10{sup -4} s{sup -1}, and h = 1.3 x 10{sup -4} and 8.5 x 10{sup -4} s{sup -1}, in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 {sup o}C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O{sub 2} does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O{sub 2}-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  2. Electroacupuncture pretreatment attenuates cerebral ischemic injury through α7 nicotinic acetylcholine receptor-mediated inhibition of high-mobility group box 1 release in rats

    Directory of Open Access Journals (Sweden)

    Wang Qiang

    2012-01-01

    Full Text Available Abstract Background We have previously reported that electroacupuncture (EA pretreatment induced tolerance against cerebral ischemic injury, but the mechanisms underlying this effect of EA are unknown. In this study, we assessed the effect of EA pretreatment on the expression of α7 nicotinic acetylcholine receptors (α7nAChR, using the ischemia-reperfusion model of focal cerebral ischemia in rats. Further, we investigated the role of high mobility group box 1 (HMGB1 in neuroprotection mediated by the α7nAChR and EA. Methods Rats were treated with EA at the acupoint "Baihui (GV 20" 24 h before focal cerebral ischemia which was induced for 120 min by middle cerebral artery occlusion. Neurobehavioral scores, infarction volumes, neuronal apoptosis, and HMGB1 levels were evaluated after reperfusion. The α7nAChR agonist PHA-543613 and the antagonist α-bungarotoxin (α-BGT were used to investigate the role of the α7nAChR in mediating neuroprotective effects. The roles of the α7nAChR and HMGB1 release in neuroprotection were further tested in neuronal cultures exposed to oxygen and glucose deprivation (OGD. Results Our results showed that the expression of α7nAChR was significantly decreased after reperfusion. EA pretreatment prevented the reduction in neuronal expression of α7nAChR after reperfusion in the ischemic penumbra. Pretreatment with PHA-543613 afforded neuroprotective effects against ischemic damage. Moreover, EA pretreatment reduced infarct volume, improved neurological outcome, inhibited neuronal apoptosis and HMGB1 release following reperfusion, and the beneficial effects were attenuated by α-BGT. The HMGB1 levels in plasma and the penumbral brain tissue were correlated with the number of apoptotic neurons in the ischemic penumbra. Furthermore, OGD in cultured neurons triggered HMGB1 release into the culture medium, and this effect was efficiently suppressed by PHA-543,613. Pretreatment with α-BGT reversed the inhibitory effect

  3. Nicotine-Mediated Regulation of Nicotinic Acetylcholine Receptors in Non-Small Cell Lung Adenocarcinoma by E2F1 and STAT1 Transcription Factors.

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    Courtney Schaal

    Full Text Available Cigarette smoking is the major risk factor for non-small cell lung cancer (NSCLC, which accounts for 80% of all lung cancers. Nicotine, the addictive component of tobacco smoke, can induce proliferation, migration, invasion, epithelial-mesenchymal transition (EMT, angiogenesis, and survival in NSCLC cell lines, as well as growth and metastasis of NSCLC in mice. This nicotine-mediated tumor progression is facilitated through activation of nicotinic acetylcholine receptors (nAChRs, specifically the α7 subunit; however, how the α7 nAChR gene is regulated in lung adenocarcinoma is not fully clear. Here we demonstrate that the α7 nAChR gene promoter is differentially regulated by E2F and STAT transcription factors through a competitive interplay; E2F1 induces the promoter, while STAT transcription factors repress it by binding to an overlapping site at a region -294 through -463bp upstream of the transcription start site. Treatment of cells with nicotine induced the mRNA and protein levels of α7 nAChR; this could be abrogated by treatment with inhibitors targeting Src, PI3K, MEK, α7 nAChR, CDK4/6 or a disruptor of the Rb-Raf-1 interaction. Further, nicotine-mediated induction of α7 nAChR was reduced when E2F1 was depleted and in contrast elevated when STAT1 was depleted by siRNAs. Interestingly, extracts from e-cigarettes, which have recently emerged as healthier alternatives to traditional cigarette smoking, can also induce α7 nAChR expression in a manner similar to nicotine. These results suggest an autoregulatory feed-forward loop that induces the levels of α7 nAChR upon exposure to nicotine, which enhances the strength of the signal. It can be imagined that such an induction of α7 nAChR contributes to the tumor-promoting functions of nicotine.

  4. Antimicrobial activity of human prion protein is mediated by its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Mukesh Pasupuleti

    Full Text Available BACKGROUND: Cellular prion-related protein (PrP(c is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

  5. Transformation of human liver L-02 cells mediated by stable HBx transfection

    Institute of Scientific and Technical Information of China (English)

    Wei-ying ZHANG; Na CAI; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To explore the mechanism of hepatocarcinogenesis associated with the hepatitis B virus X protein (HBx), we investigated the role of HBx in transformation using human liver L-02 cells stably transfected with HBx as a model.Methods: Plasmids encoding HBx were stably transfected into immortalized human liver L-02 cells and rodent fibroblast NIH/3T3 cells. The expression of alfa-fetoprotein (AFP), c-Myc, HBx, and survivin in the engineered cells was examined by Western blotting. The malignant phenotype of the cells was demonstrated by anchorage-independent colony formation and tumor formation in nude mice. RNA interference assays, Western blotting, luciferase reporter gene assays and flow cytometry analysis were performed. The number of centrosomes in the L-O2-X cells was determined by Y-tubulin immunostaining. The effect of HBx on the transcriptional activity of human telomerase reverse transcriptase (hTERT) and hTERT activity in L-02-X cells and/or 3T3-X cells was detected by the luciferase reporter gene assay and telomerase repeat amplification protocol (TRAP).Results: Stable HBx transfection resulted in a malignant phenotype in the engineered cells in vivo and in vitro. Meanwhile, HBx was able to increase the transcription of the NF-κB, AP-1, and survivin genes and to upregulate the expression levels of c-Myc and survivin.Abnormal centrosome duplication and activated hTERT were responsible for the transformation.Conclusion: Stable HBx transfection leads to genomic instability of host cells, which is responsible for hepatocarcinogenesis; mean-while, transactivation by the HBx protein contributes to the development of hepatocellular carcinoma (HCC). The L-02-X cell line is an ideal model for investigating the mechanism of HBx-mediated transformation.

  6. Visfatin as a novel mediator released by inflamed human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Tania Romacho

    Full Text Available BACKGROUND: Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. METHODS AND RESULTS: Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC. However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL-1β (1 to 10 ng/mL, tumor necrosis factor-α (1 to 10 ng/mL or angiotensin II (10 pmol/L to 1 μmol/L markedly enhanced intracellular visfatin content. Using IL-1β (10 ng/mL; 18 h, it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose polymerase-1 activation. Moreover, IL-1β modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1β-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1β promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. CONCLUSIONS: Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation.

  7. Bradykinin and adenosine receptors mediate desflurane induced postconditioning in human myocardium: role of reactive oxygen species

    Directory of Open Access Journals (Sweden)

    Gérard Jean-Louis

    2010-07-01

    Full Text Available Abstract Background Desflurane during early reperfusion has been shown to postcondition human myocardium, in vitro. We investigated the role of adenosine and bradykinin receptors, and generation of radical oxygen species in desflurane-induced postconditioning in human myocardium. Methods We recorded isometric contraction of human right atrial trabeculae hanged in an oxygenated Tyrode's solution (34 degrees Celsius, stimulation frequency 1 Hz. After a 30-min hypoxic period, desflurane 6% was administered during the first 5 min of reoxygenation. Desflurane was administered alone or with pretreatment of N-mercaptopropionylglycine, a reactive oxygen species scavenger, 8-(p-Sulfophenyltheophylline, an adenosine receptor antagonist, HOE140, a selective B2 bradykinin receptor antagonist. In separate groups, adenosine and bradykinin were administered during the first minutes of reoxygenation alone or in presence of N-mercaptopropionylglycine. The force of contraction of trabeculae was recorded continuously. Developed force at the end of a 60-min reoxygenation period was compared (mean ± standard deviation between the groups by a variance analysis and post hoc test. Results Desflurane 6% (84 ± 6% of baseline enhanced the recovery of force after 60-min of reoxygenation as compared to control group (51 ± 8% of baseline, P N-mercaptopropionylglycine (54 ± 3% of baseline, 8-(p-Sulfophenyltheophylline (62 ± 9% of baseline, HOE140 (58 ± 6% of baseline abolished desflurane-induced postconditioning. Adenosine (80 ± 9% of baseline and bradykinin (83 ± 4% of baseline induced postconditioning (P vs control, N-mercaptopropionylglycine abolished the beneficial effects of adenosine and bradykinin (54 ± 8 and 58 ± 5% of baseline, respectively. Conclusions In vitro, desflurane-induced postconditioning depends on reactive oxygen species production, activation of adenosine and bradykinin B2 receptors. And, the cardioprotective effect of adenosine and bradykinin

  8. Rapid effects of phytoestrogens on human colonic smooth muscle are mediated by oestrogen receptor beta.

    LENUS (Irish Health Repository)

    Hogan, A M

    2012-02-01

    Epidemiological studies have correlated consumption of dietary phytoestrogens with beneficial effects on colon, breast and prostate cancers. Genomic and non-genomic mechanisms are responsible for anti-carcinogenic effects but, until now, the effect on human colon was assumed to be passive and remote. No direct effect on human colonic smooth muscle has previously been described. Institutional research board approval was granted. Histologically normal colon was obtained from the proximal resection margin of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended under 1g of tension in organ baths containing oxygenated Krebs solution at 37 degrees C. After an equilibration period, tissues were exposed to diarylpropionitrile (DPN) (ER beta agonist) and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (ER alpha agonist) or to the synthetic phytoestrogen compounds genistein (n=8), daidzein (n=8), fisetin (n=8) and quercetin (n=8) in the presence or absence of fulvestrant (oestrogen receptor antagonist). Mechanism of action was investigated by inhibition of downstream pathways. The cholinergic agonist carbachol was used to induce contractile activity. Tension was recorded isometrically. Phytoestrogens inhibit carbachol-induced colonic contractility. In keeping with a non-genomic, rapid onset direct action, the effect was within minutes, reversible and similar to previously described actions of 17 beta oestradiol. No effect was seen in the presence of fulvestrant indicating receptor modulation. While the DPN exerted inhibitory effects, PPT did not. The effect appears to be reliant on a p38\\/mitogen activated protein kinase mediated induction of nitric oxide production in colonic smooth muscle. The present data set provides the first description of a direct effect of genistein, daidzein, fisetin and quercetin on human colonic smooth muscle. The presence of ER in colonic smooth muscle has been functionally proven and the beta

  9. Human-Mediated Marine Dispersal Influences the Population Structure of Aedes aegypti in the Philippine Archipelago.

    Directory of Open Access Journals (Sweden)

    Eugenio Fonzi

    Full Text Available Dengue virus (DENV is an extraordinary health burden on global scale, but still lacks effective vaccine. The Philippines is endemic for dengue fever, but massive employment of insecticides favored the development of resistance mutations in its major vector, Aedes aegypti. Alternative vector control strategies consist in releasing artificially modified mosquitos in the wild, but knowledge on their dispersal ability is necessary for a successful implementation. Despite being documented that Ae. aegypti can be passively transported for long distances, no study to date has been aimed at understanding whether human marine transportation can substantially shape the migration patterns of this mosquito. With thousands of islands connected by a dense network of ships, the Philippines is an ideal environment to fill this knowledge gap.Larvae of Ae. aegypti from 15 seaports in seven major islands of central-western Philippines were collected and genotyped at seven microsatellite loci. Low genetic structure and considerable gene flow was found in the area. Univariate and multivariate regression analyses suggested that anthropic factors (specifically the amount of processed cargo and human population density can explain the observed population structure, while geographical distance was not correlated. Interestingly, cargo shipments seem to be more efficient than passenger ships in transporting Ae. aegypti. Bayesian clustering confirmed that Ae. aegypti from busy ports are more genetically similar, while populations from idle ports are relatively structured, regardless of the geographical distance that separates them.The results confirmed the pivotal role of marine human-mediated long-range dispersal in determining the population structure of Ae. aegypti. Hopefully corroborated by further research, the present findings could assist the design of more effective vector control strategies.

  10. Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

    Science.gov (United States)

    Bridgen, D T; Gilchrist, C L; Richardson, W J; Isaacs, R E; Brown, C R; Yang, K L; Chen, J; Setton, L A

    2013-10-01

    The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

  11. The molecular mechanisms of Curcuma Wenyujin extract-mediated inhibitory effects on human esophageal carcinoma cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    景钊

    2012-01-01

    Objective To study the molecular mechanisms of Curcuma Wenyujin extract-mediated inhibitory effects on human esophageal carcinoma cells. Methods The Curcuma Wenyujin extract was obtained by supercritical carbon dioxide extraction. TE-1 cells were divided into 4 groups after adherence.

  12. How the Organizational Learning Process Mediates the Impact of Strategic Human Resource Management Practices on Performance in Korean Organizations

    Science.gov (United States)

    Cho, Sei Hyoung; Song, Ji Hoon; Yun, Suk Chun; Lee, Cheol Ki

    2013-01-01

    The primary purpose of this research is to examine the structural relationships among several workplace-related constructs, including strategic human resource management (HRM) practices, organizational learning processes, and performance improvement in the Korean business context. More specifically, the research examined the mediating effect of…

  13. How the Organizational Learning Process Mediates the Impact of Strategic Human Resource Management Practices on Performance in Korean Organizations

    Science.gov (United States)

    Cho, Sei Hyoung; Song, Ji Hoon; Yun, Suk Chun; Lee, Cheol Ki

    2013-01-01

    The primary purpose of this research is to examine the structural relationships among several workplace-related constructs, including strategic human resource management (HRM) practices, organizational learning processes, and performance improvement in the Korean business context. More specifically, the research examined the mediating effect of…

  14. The Impact of Bundled High Performance Human Resource Practices on Intention to Leave: Mediating Role of Emotional Exhaustion

    Science.gov (United States)

    Jyoti, Jeevan; Rani, Roomi; Gandotra, Rupali

    2015-01-01

    Purpose: The purpose of this paper is to examine the mediating effect of emotional exhaustion (EE) in between bundled high-performance human resource practices (HPHRPs) and intention to leave (ITL) in the education sector. Design/methodology/approach: A survey questionnaire method was used to collect data from a sample of 514 teachers working in…

  15. The Impact of Bundled High Performance Human Resource Practices on Intention to Leave: Mediating Role of Emotional Exhaustion

    Science.gov (United States)

    Jyoti, Jeevan; Rani, Roomi; Gandotra, Rupali

    2015-01-01

    Purpose: The purpose of this paper is to examine the mediating effect of emotional exhaustion (EE) in between bundled high-performance human resource practices (HPHRPs) and intention to leave (ITL) in the education sector. Design/methodology/approach: A survey questionnaire method was used to collect data from a sample of 514 teachers working in…

  16. Adhesion, invasion, and agglutination mediated by two trimeric autotransporters in the human uropathogen Proteus mirabilis.

    Science.gov (United States)

    Alamuri, Praveen; Löwer, Martin; Hiss, Jan A; Himpsl, Stephanie D; Schneider, Gisbert; Mobley, Harry L T

    2010-11-01

    Fimbriae of the human uropathogen Proteus mirabilis are the only characterized surface proteins that contribute to its virulence by mediating adhesion and invasion of the uroepithelia. PMI2122 (AipA) and PMI2575 (TaaP) are annotated in the genome of strain HI4320 as trimeric autotransporters with "adhesin-like" and "agglutinating adhesin-like" properties, respectively. The C-terminal 62 amino acids (aa) in AipA and 76 aa in TaaP are homologous to the translocator domains of YadA from Yersinia enterocolitica and Hia from Haemophilus influenzae. Comparative protein modeling using the Hia three-dimensional structure as a template predicted that each of these domains would contain four antiparallel beta sheets and that they formed homotrimers. Recombinant AipA and TaaP were seen as ∼28 kDa and ∼78 kDa, respectively, in Escherichia coli, and each also formed high-molecular-weight homotrimers, thus supporting this model. E. coli synthesizing AipA or TaaP bound to extracellular matrix proteins with a 10- to 60-fold-higher level of affinity than the control strain. Inactivation of aipA in P. mirabilis strains significantly (P < 0.01) reduced the mutants' ability to adhere to or invade HEK293 cell monolayers, and the functions were restored upon complementation. A 51-aa-long invasin region in the AipA passenger domain was required for this function. E. coli expressing TaaP mediated autoagglutination, and a taaP mutant of P. mirabilis showed significantly (P < 0.05) more reduced aggregation than HI4320. Gly-247 in AipA and Gly-708 in TaaP were indispensable for trimerization and activity. AipA and TaaP individually offered advantages to P. mirabilis in a murine model. This is the first report characterizing trimeric autotransporters in P. mirabilis as afimbrial surface adhesins and autoagglutinins.

  17. MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts.

    Science.gov (United States)

    Liu, Haijun; Dai, Xiaoniu; Cheng, Yusi; Fang, Shencun; Zhang, Yingming; Wang, Xingang; Zhang, Wei; Liao, Hong; Yao, Honghong; Chao, Jie

    2016-01-15

    Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

  18. Copper Oxide Nanoparticles Induced Mitochondria Mediated Apoptosis in Human Hepatocarcinoma Cells

    Science.gov (United States)

    Siddiqui, Maqsood A.; Alhadlaq, Hisham A.; Ahmad, Javed; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed; Ahamed, Maqusood

    2013-01-01

    Copper oxide nanoparticles (CuO NPs) are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2–50 µg/ml) were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at in vitro level. Underlying mechanism(s) of apoptosis due to CuO NPs exposure should be further invested at in vivo level. PMID:23940521

  19. Hypericin-mediated photooxidative damage of α-crystallin in human lens epithelial cells.

    Science.gov (United States)

    Ehrenshaft, Marilyn; Roberts, Joan E; Mason, Ronald P

    2013-07-01

    St. John's wort (Hypericum perforatum), a perennial herb native to Europe, is widely used for and seems to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and the UVA range, is considered to be one of the bioactive ingredients of St. John's wort, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. α-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescence confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths St. John's wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage.

  20. A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes.

    Science.gov (United States)

    Brambilla, R; Ceruti, S; Malorni, W; Cattabeni, F; Abbracchio, M P

    2000-07-01

    In astrocytic cultures maintained in vitro, a brief challenge with the ATP analog alpha,beta methyleneATP (alpha,betameATP) results, 3 days later, in marked elongation of astrocytic processes, an event that resembles the astrocytic hypertrophy known to occur in vivo during reactive astrogliosis. alpha,beta meATP-induced effects were observed in primary astrocytes obtained from both rat striatum and cortex (a brain area highly involved in chronic neurodegenerative pathologies), as well as in human astrocytoma cells (ADF cells). Purine-induced gliosis could be reversed by the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS), but not by oxidized ATP (an antagonist of the P2X(7) receptor), in line with previous studies of our laboratory suggesting the involvement of a P2Y receptor subtype. Induction of reactive gliosis was preceded by increased expression of cyclooxygenase-2 (COX-2), an enzyme whose excessive activation has been implicated in both acute and chronic neurodegenerative diseases. The selective COX-2 inhibitor NS-398 prevented both purine-induced astrogliosis and the associated COX-2 induction, suggesting that inhibition of the transcription of the COX-2 gene may also contribute to the anti-inflammatory properties of this agent. Significant blockade of both alpha,beta meATP-mediated reactive gliosis and COX-2 induction was also observed with PPADS. These data suggest that COX-2 mediates P2Y receptor-induced reactive astrogliosis, and that antagonists selective for this receptor subtype may represent a novel class of anti-inflammatory agents of potential interest in acute and chronic neurological disorders characterized by an inflammatory component and reactive gliosis.

  1. TFF3 mediated induction of VEGF via hypoxia in human gastric cancer SGC-7901 cells.

    Science.gov (United States)

    Guleng, Bayasi; Han, Jia; Yang, Jin-Qiu; Huang, Qing-Wen; Huang, Jian-Kun; Yang, Xiao-Ning; Liu, Jing-Jing; Ren, Jian-Lin

    2012-04-01

    Increasing evidence indicates that in gastric epithelial cells, induction of TFF3 by hypoxia is mediated by HIF-1. Since VEGF is one of the most important angiogenic factors on cancer progression, we have started to investigate the possible link among HIF-1α, VEGF, and TFF3 in gastric cancer cells. We induced the hypoxic condition in SGC-7901cells using hypoxia-mimetic agent of CoCI2. SGC7901 cells were transfected with pcPUR + U6 plasmid carrying RNAi targeted to human TFF3 and selected puromycin-resistant pools to establish the stable knockdown of TFF3 cells. Our results showed the induction of HIF-1a via hypoxia and consequences of increased expressions of the TFF3 and VEGF in gastric cancer SGC-7901 cells. Overexpression of TFF3 upregulated the mRNA expressions of VEGF and HIF-1a induced by hypoxia, and stable knockdown of TFF3 impaired the mRNA upregulations of VEGF and HIF-1a induced by hypoxia. Furthermore, knockdown of TFF3 reduced the VEGF protein secretion: as VEGF secretion was increased time dependent manner in response to the hypoxia induction in TFF3-WT cells; however, VEGF production was significantly decreased in TFF3-KD cells (621 ± 89 vs. 264 ± 73 at 6 h and 969 ± 97 vs. 508 ± 69 at 12 h, P TFF3 mediated regulation of VEGF expression induced by hypoxia, and implicated that TFF3 might be applied as a potential anti-angiogenic target for treatment of gastric cancer.

  2. Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepatocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Maqsood A Siddiqui

    Full Text Available Copper oxide nanoparticles (CuO NPs are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2-50 µg/ml were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at in vitro level. Underlying mechanism(s of apoptosis due to CuO NPs exposure should be further invested at in vivo level.

  3. Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepatocarcinoma cells.

    Science.gov (United States)

    Siddiqui, Maqsood A; Alhadlaq, Hisham A; Ahmad, Javed; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Ahamed, Maqusood

    2013-01-01

    Copper oxide nanoparticles (CuO NPs) are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2-50 µg/ml) were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at in vitro level. Underlying mechanism(s) of apoptosis due to CuO NPs exposure should be further invested at in vivo level.

  4. Human Papilloma Virus 16 E6 RNA Interference Enhances Cisplatin and Death Receptor-Mediated Apoptosis in Human Cervical Carcinoma Cells

    NARCIS (Netherlands)

    Tan, Shinta; Hougardy, Brigitte M. T.; Meersma, Gert J.; Schaap, Bessel; de Vries, Elisabeth G. E.; van der Zee, Ate G. J.; de Jong, Steven

    2012-01-01

    In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfer

  5. SPOC1-Mediated Antiviral Host Cell Response Is Antagonized Early in Human Adenovirus Type 5 Infection

    Science.gov (United States)

    Schreiner, Sabrina; Kinkley, Sarah; Bürck, Carolin; Mund, Andreas; Wimmer, Peter; Schubert, Tobias; Groitl, Peter; Will, Hans; Dobner, Thomas

    2013-01-01

    Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24–48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming

  6. Hydrogen sulfide mediates the anti-survival effect of sulforaphane on human prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Pei, Yanxi [Department of Biology, Lakehead University, Thunder Bay (Canada); College of Life Science, Shanxi University, Taiyuan (China); Wu, Bo [Department of Biology, Lakehead University, Thunder Bay (Canada); Department of Pathophysiology, Harbin Medical University, Harbin (China); Cao, Qiuhui [Department of Biology, Lakehead University, Thunder Bay (Canada); Wu, Lingyun [Department of Pathophysiology, Harbin Medical University, Harbin (China); Department of Pharmacology, University of Saskatchewan, Saskatoon (Canada); Yang, Guangdong, E-mail: gyang@lakeheadu.ca [The School of Kinesiology, Lakehead University, Thunder Bay (Canada)

    2011-12-15

    Hydrogen sulfide (H{sub 2}S) is a novel gasotransmitter that regulates cell proliferation and other cellular functions. Sulforaphane (SFN) is a sulfur-containing compound that exhibits anticancer properties, and young sprouts of broccoli are particularly rich in SFN. There is consistent epidemiological evidence that the consumption of sulfur-containing vegetables, such as garlic and cruciferous vegetables, may help reduce the occurrence of prostate cancer. Here we found that a large amount of H{sub 2}S is released when SFN is added into cell culture medium or mixed with mouse liver homogenates, respectively. Both SFN and NaHS (a H{sub 2}S donor) decreased the viability of PC-3 cells (a human prostate cancer cell line) in a dose-dependent manner, and supplement of methemoglobin or oxidized glutathione (two H{sub 2}S scavengers) reversed SFN-reduced cell viability. We further found both cystathionine gamma-lyase (CSE) and cystathionine beta-synthase are expressed in PC-3 cells and mouse prostate tissues. H{sub 2}S production in prostate tissues from CSE knockout mice was only 20% of that from wild-type mice, suggesting CSE is a major H{sub 2}S-producing enzyme in prostate. CSE overexpression enhanced H{sub 2}S production and inhibited cell viability in PC-3 cells. In addition, both SFN and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK). Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H{sub 2}S- or SFN-reduced viability of PC-3 cells. Our results demonstrated that H{sub 2}S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells, which suggests that H{sub 2}S-releasing diet or drug might be beneficial in the treatment of prostate cancer. Highlights: Black-Right-Pointing-Pointer A large amount of H{sub 2}S is released from sulforaphane. Black-Right-Pointing-Pointer H{sub 2}S mediates the anti-survival effect of

  7. The nicotinic alpha7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-beta1-42

    DEFF Research Database (Denmark)

    Søderman, Andreas; Thomsen, Morten Skøtt; Hansen, Henrik H;

    2008-01-01

    Recent studies have demonstrated that amyloid-beta1-42 (Abeta1-42) binds to the nicotinergic alpha7 acetylcholine receptor (alpha7 nAChR) and that the application of Abeta1-42 to cells inhibits the function of the alpha7 nAChR. The in vivo consequences of the pharmacological activation of the alpha...... through the use of co-immunoprecipitation that human Abeta-immunoreactive peptides bind to mice alpha7 nAChR in vivo. Agonists of the alpha7 nAChR improve memory and attentional properties and increase immediate early gene expression in the prefrontal cortex and the nucleus accumbens. We show that acute...

  8. Prioritization of capacities for the elimination of dog-mediated human rabies in the Americas: building the framework.

    Science.gov (United States)

    Del Rio Vilas, Victor J; Burgeño, Adamelia; Montibeller, Gilberto; Clavijo, Alfonso; Vigilato, Marco Antonio; Cosivi, Ottorino

    2013-10-01

    The region of the Americas pledged to eliminate dog-transmitted human rabies by 2015. After 30 years of sustained efforts, regional elimination appears possible as dog-mediated human rabies cases are at an all-time low, and a number of countries and territories have already eliminated the disease. In this setting, there is an opportunity to generate a framework to support countries strategies in the achievement and maintenance of rabies-free status (RFS). To this end, we describe the development of a multi-criteria decision analysis (MCDA) model to help the evaluation of rabies programmes and the identification of the best investment strategy for countries and territories to improve and efficiently maintain their rabies status. The model contemplates human and animal related capacities, six in each area, to comprehensively assess the wide scope of rabies programmes. An initial elicitation of expert opinion of values and weights for the MCDA model was performed via a web-based questionnaire. Even at this pilot stage, the model produces comparable capacity-scores, and overall (combined for public and animal health areas) as well as area-specific investment strategies. The model is being developed by the Pan American Health Organization (PAHO) as part of the regional efforts towards dog-mediated human rabies elimination and will be presented to the countries for review, refinement, contextualization, and testing. The aspiration is that countries use the model to identify the best allocation of resources towards the elimination of dog-mediated human rabies.

  9. [Ethnic evaluation of hospital humanization by the users of the Brazilian Unified Health System and their mediators].

    Science.gov (United States)

    Gomes, Annatália Meneses de Amorim; Moura, Escolástica Rejane Ferreira; Nations, Marilyn Kay; Alves, Maria do Socorro Costa Feitosa

    2008-12-01

    This study was aimed at evaluating the humanization of hospital care ethnically, as perceived by the hospitalized patients. Data were collected from 13 inpatients from January to July/2005 in a public hospital in Fortaleza, Ceará, Brazil, according to the patient's circuit. This analysis yielded the following categories: ethnic evaluation of the hospital structure and dynamics, hospital and professional image, human and technical competence. The subjects used multiple aspects to soften their opinions, unveiling factors named mediators of the ethnical evaluation. Such aspects were categorized into: conditions of the interview, socio-economic status, user's personality and religiousness, ironic speech, somber diagnosis and necessities met, and prior hospitalization experiences. The elements revealed by the patients are significant in order to encourage professionals and mediators to mobilize towards humanizing changes, including the user as a social and critical ethnic evaluator.

  10. The PPARα-Humanized Mouse: A Model to Investigate Species Differences in Liver Toxicity Mediated by PPARα

    OpenAIRE

    Yang, Qian; Nagano, Tomokazu; Shah, Yatrik; Cheung, Connie; ITO, SHINJI; Gonzalez, Frank J.

    2007-01-01

    To determine the impact of the species difference between rodents and humans in response to peroxisome proliferators (PPs) mediated by peroxisome proliferator–activated receptor (PPAR)α, PPARα-humanized transgenic mice were generated using a P1 phage artificial chromosome (PAC) genomic clone bred onto a pparα-null mouse background, designated hPPARαPAC. In hPPARαPAC mice, the human PPARα gene is expressed in tissues with high fatty acid catabolism and induced upon fasting, similar to mouse PP...

  11. Glucocorticoids Suppress CCR9-Mediated Chemotaxis, Calcium Flux, and Adhesion to MAdCAM-1 in Human T Cells.

    Science.gov (United States)

    Wendt, Emily; White, Gemma E; Ferry, Helen; Huhn, Michael; Greaves, David R; Keshav, Satish

    2016-05-01

    CCR9 expressed on T lymphocytes mediates migration to the small intestine in response to a gradient of CCL25. CCL25-stimulated activation of α4β7 integrin promotes cell adherence to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed by vascular endothelial cells of the intestine, further mediating gut-specific homing. Inflammatory bowel disease is a chronic inflammatory condition that primarily affects the gastrointestinal tract and is characterized by leukocyte infiltration. Glucocorticoids (GCs) are widely used to treat inflammatory bowel disease but their effect on intestinal leukocyte homing is not well understood. We investigated the effect of GCs on the gut-specific chemokine receptor pair, CCR9 and CCL25. Using human peripheral blood-derived T lymphocytes enriched for CCR9 by cell sorting or culturing with all-trans retinoic acid, we measured chemotaxis, intracellular calcium flux, and α4β7-mediated cell adhesion to plate-bound MAdCAM-1. Dexamethasone (DEX), a specific GC receptor agonist, significantly reduced CCR9-mediated chemotaxis and adhesion to MAdCAM-1 without affecting CCR9 surface expression. In contrast, in the same cells, DEX increased CXCR4 surface expression and CXCL12-mediated signaling and downstream functions. The effects of DEX on human primary T cells were reversed by the GC receptor antagonist mifepristone. These results demonstrate that GCs suppress CCR9-mediated chemotaxis, intracellular calcium flux, and α4β7-mediated cell adhesion in vitro, and these effects could contribute to the efficacy of GCs in treating intestinal inflammation in vivo.

  12. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans.

    Science.gov (United States)

    Schroeter, Hagen; Heiss, Christian; Balzer, Jan; Kleinbongard, Petra; Keen, Carl L; Hollenberg, Norman K; Sies, Helmut; Kwik-Uribe, Catherine; Schmitz, Harold H; Kelm, Malte

    2006-01-24

    Epidemiological and medical anthropological investigations suggest that flavanol-rich foods exert cardiovascular health benefits. Endothelial dysfunction, a prognostically relevant key event in atherosclerosis, is characterized by a decreased bioactivity of nitric oxide (NO) and impaired flow-mediated vasodilation (FMD). We show in healthy male adults that the ingestion of flavanol-rich cocoa was associated with acute elevations in levels of circulating NO species, an enhanced FMD response of conduit arteries, and an augmented microcirculation. In addition, the concentrations and the chemical profiles of circulating flavanol metabolites were determined, and multivariate regression analyses identified (-)-epicatechin and its metabolite, epicatechin-7-O-glucuronide, as independent predictors of the vascular effects after flavanol-rich cocoa ingestion. A mixture of flavanols/metabolites, resembling the profile and concentration of circulating flavanol compounds in plasma after cocoa ingestion, induced a relaxation in preconstricted rabbit aortic rings ex vivo, thus mimicking acetylcholine-induced relaxations. Ex vivo flavanol-induced relaxation, as well as the in vivo increases in FMD, were abolished by inhibition of NO synthase. Oral administration of chemically pure (-)-epicatechin to humans closely emulated acute vascular effects of flavanol-rich cocoa. Finally, the concept that a chronic intake of high-flavanol diets is associated with prolonged, augmented NO synthesis is supported by data that indicate a correlation between the chronic consumption of a cocoa flavanol-rich diet and the augmented urinary excretion of NO metabolites. Collectively, our data demonstrate that the human ingestion of the flavanol (-)-epicatechin is, at least in part, causally linked to the reported vascular effects observed after the consumption of flavanol-rich cocoa.

  13. Integrin-mediated cell migration is blocked by inhibitors of human neuraminidase.

    Science.gov (United States)

    Jia, Feng; Howlader, Md Amran; Cairo, Christopher W

    2016-09-01

    Integrins are critical receptors in cell migration and adhesion. A number of mechanisms are known to regulate the function of integrins, including phosphorylation, conformational change, and cytoskeletal anchoring. We investigated whether native neuraminidase (Neu, or sialidase) enzymes which modify glycolipids could play a role in regulating integrin-mediated cell migration. Using a scratch assay, we found that exogenously added Neu3 and Neu4 activity altered rates of cell migration. We observed that Neu4 increased the rate of migration in two cell lines (HeLa, A549); while Neu3 only increased migration in HeLa cells. A bacterial neuraminidase was able to increase the rate of migration in HeLa, but not in A549 cells. Treatment of cells with complex gangliosides (GM1, GD1a, GD1b, and GT1b) resulted in decreased cell migration rates, while LacCer was able to increase rates of migration in both lines. Importantly, our results show that treatment of cells with inhibitors of native Neu enzymes had a dramatic effect on the rates of cell migration. The most potent compound tested targeted the human Neu4 isoenzyme, and was able to substantially reduce the rate of cell migration. We found that the lateral mobility of integrins was reduced by treatment of cells with Neu3, suggesting that Neu3 enzyme activity resulted in changes to integrin-co-receptor or integrin-cytoskeleton interactions. Finally, our results support the hypothesis that inhibitors of human Neu can be used to investigate mechanisms of cell migration and for the development of anti-adhesive therapies.

  14. Sodium butyrate induces DRP1-mediated mitochondrial fusion and apoptosis in human colorectal cancer cells.

    Science.gov (United States)

    Tailor, Dhanir; Hahm, Eun-Ryeong; Kale, Raosaheb K; Singh, Shivendra V; Singh, Rana P

    2014-05-01

    Sodium butyrate (NaBt) is the byproduct of anaerobic microbial fermentation inside the gastro-intestinal tract that could reach up to 20mM, and has been shown to inhibit the growth of various cancers. Herein, we evaluated its effect on mitochondrial fusion and associated induction of apoptosis in colorectal cancer cells (CRC). NaBt treatment at physiological (1-5mM) concentrations for 12 and 24h decreased the cell viability and induced G2-M phase cell cycle arrest in HCT116 (12h) and SW480 human CRC cells. This cell cycle arrest was associated with mitochondria-mediated apoptosis accompanied by a decrease in survivin and Bcl-2 expression, and generation of reactive oxygen species (ROS). Furthermore, NaBt treatment resulted in a significant decrease in the mitochondrial mass which is an indicator of mitochondrial fusion. Level of dynamin-related protein 1 (DRP1), a key regulator of mitochondrial fission and fusion where its up-regulation correlates with fission, was found to be decreased in CRC cells. Further, at early treatment time, DRP1 down-regulation was noticed in mitochondria which later became drastically reduced in both mitochondria as well as cytosol. DRP1 is activated by cyclin B1-CDK1 complex by its ser616 phosphorylation in which both cyclin B1-CDK1 complex and phospho-DRP1 (ser616) were strongly reduced by NaBt treatment. DRP1 was observed to be regulated by apoptosis as pan-caspase inhibitor showing rescue from NaBt-induced apoptosis also caused the reversal of DRP1 to the normal level as in control proliferating cells. Together, these findings suggest that NaBt can modulate mitochondrial fission and fusion by regulating the level of DRP1 and induce cell cycle arrest and apoptosis in human CRC cells.

  15. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  16. GROWTH INHIBITION OF HUMAN LARYNGEAL CANCER CELL WITH THE ADENOVIRUS-MEDIATED p53 GENE

    Institute of Scientific and Technical Information of China (English)

    WANG Qi; HAN De-min; WANG Wen-ge; WU Zu-ze; ZHANG Wei

    1999-01-01

    Objective: In most laryngeal cancers, the function of p53 gene is down regulated. To explore the potential use of p53 in gene therapy of laryngeal cancer, by introducing wild-type p53 into laryngeal cancer cell line via a recombinant adenoviral vector, Ad5CMV-p53 and analyzing its effects on cell and tumor growth. Methods: A human laryngeal cancer cell line Hep-2 was used.Recombinant cytomegalovirus-promoted adenoviruses containing human wild-type p53 cDNA was transiently introduced into Hep-2 line. The growth suppression of the Hep-2 cells and established s.c. squamous carcinoma model was examined. The p53 protein expression was detected using immunohistochemical analysis. Results: The transduction efficiencies of Hep-2 cell line were 100% at a multiplicity of 100 or greater. The p53 protein expression peaked on day 2 after infection and lasted far 5 days. In vitro growth assays revealed cell death following Ad5CMV-p53 infected. In vivo studies, Ad5CMV-p53 inhibited the tumorigenicity of Hep-2 cell, and in nude mice with established s.c. squamous carcinoma nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. Conclusion: Adenovirus-mediated antitumor therapy carrying the p53 gene is an efficient method to inhibit laryngeal cancer growth. Transfection of laryngeal cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential novel approach to the therapy of laryngeal cancer.

  17. Reduction of the invasiveness of human glioma cells by ALA-mediated photodynamic therapy

    Science.gov (United States)

    Hirschberg, Henry; Sun, Chung-Ho; Madsen, Steen

    2006-02-01

    Introduction: High grade gliomas are characterised by rapid and invasive growth, that cause massive tissue destruction at both the tumour- brain boarder as well as in regions remote from the tumor core. Eradication or inhibition of infiltrating glioma cells poses a significant clinical challenge that is unlikely to be solved using conventional treatment regimens consisting of ionizing radiation and chemotherapeutic agents. In this study we evaluated the effects of ALA mediated photodynamic therapy (PDT) on the invesivness of human glioma cells migrating from implanted multicell tumor spheroids. Materials and method 3-400nm diameter tumor spheroids, derived from the human glioma cell line ACBT, were implanted into a gel matrix of collagen type I. 24 hours following implantation there was a significant invasion of the surrounding gel by individual tumor cells to an average distance of 400nm. The cultures were incubated in increasing concentrations of ALA (10-1000 ug/ml) for four hours and then exposed to 635nm laser light in a titration of both fluence level and fluence rate. Results Fluences of 25J/cm2 were clearly cytotoxic for both the infiltrating cells as well as the spheroids at all ALA concentrations. Fluence levels of 6J did not stop the spheroid growth or prove cytotoxic to the glioma cells that had previously migrated into the gel, in a majority of cultures but inhibited further migration of the cells by 80-90% compared to control. Conclusion: Measurement of cell survival and cell proliferation indices seemed to indicate a direct migratory inhibition effect on the invading cells and not cytotoxicity as the most likely mechanism for this observation.

  18. Plasmid-mediated quinolone resistance; interactions between human, animal and environmental ecologies

    Directory of Open Access Journals (Sweden)

    Laurent ePOIREL

    2012-02-01

    Full Text Available Resistance to quinolones and fluoroquinolones is being increasingly reported among human but also veterinary isolates during the last two to three decades, very likely as a consequence of the large clinical usage of those antibiotics. Even if the principle mechanisms of resistance to quinolones are chromosome-encoded, due to modifications of molecular targets (DNA gyrase and topoisomerase IV, decreased outer-membrane permeability (porin defect and overexpression of naturally-occurring efflux, the emergence of plasmid-mediated quinolone resistance (PMQR has been reported since 1998. Although these PMQR determinants confer low-level resistance to quinolones and/or fluoroquinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms. Different transferable mechanisms have been identified, corresponding to the production of Qnr proteins, of the aminoglycoside acetyltransferase AAC(6’-Ib-cr, or of the QepA-type or OqxAB-type efflux pumps. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase from quinolone inhibition (mostly nalidixic acid. The AAC(6’-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. Finally, the QepA and OqxAB efflux pumps extrude fluoroquinolones from the bacterial cell. A series of studies have identified the environment to be a reservoir of PMQR genes, with farm animals and aquatic habitats being significantly involved. In addition, the origin of the qnr genes has been identified, corresponding to the waterborne species Shewanella sp. Altogether, the recent observations suggest that the aquatic environment might constitute the original source of PMQR genes, that would secondly spread among animal or human isolates.

  19. Glucocorticoid-mediated effects on metabolism are reversed by targeting 11 beta hydroxysteroid dehydrogenase type 1 in human skeletal muscle.

    Science.gov (United States)

    Salehzadeh, Firoozeh; Al-Khalili, Lubna; Kulkarni, Sameer S; Wang, Minghan; Lönnqvist, Fredrik; Krook, Anna

    2009-03-01

    Adipose tissue and liver play important roles in mediating the metabolic actions of glucocorticoids. However, the effects of glucocorticoids on glucose and lipid metabolism in skeletal muscle are not understood completely. Intracellular glucocorticoid action is dependent on 11 beta-hydroxysteroid dehydrogenase 1 (HSD1), an enzyme that converts cortisone to active cortisol. We investigated the direct role of HSD1 in cultured primary human skeletal muscle cells using siRNA and pharmacological inhibitors of the enzyme. Primary human skeletal muscle cells were cultured in the presence of 0.5 microM cortisone or 0.5 microM cortisol for eight days. siRNA was utilized to reduce expression of either HSD1 or pyruvate dehydrogenase kinase (PDK) 4. Effects of pharmacological inhibitors of HSD1 were also studied. Exposure to cortisone or cortisol decreased basal glucose uptake and glucose incorporation into glycogen, but was without effect on the insulin-stimulated response. Glucocorticoid exposure increased palmitate oxidation, as well as the expression of PDK4. siRNA-mediated reduction or pharmacological inhibition of HSD1 prevented the effects of cortisone, but not cortisol, on metabolic responses. siRNA-mediated reduction of PDK4 prevented the effect of cortisol to attenuate glycogen synthesis. Targeted reduction or pharmacological inhibition of HSD1 in primary human skeletal muscle cells prevents the effects of cortisone, but not cortisol, on glucose metabolism and palmitate oxidation. Furthermore, the glucocorticoid-mediated reductions in glucose metabolism are dependent on PDK4.

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

    Science.gov (United States)

    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.

  1. Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

    Directory of Open Access Journals (Sweden)

    Kun Liu

    2012-05-01

    Full Text Available Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA. Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

  2. Human dermis separation via ultra-short pulsed laser plasma-mediated ablation

    Energy Technology Data Exchange (ETDEWEB)

    Huang Huan; Guo Zhixiong, E-mail: guo@jove.rutgers.ed [Department of Mechanical and Aerospace Engineering, Rutgers, State University of New Jersey, Piscataway, NJ 08854 (United States)

    2009-08-21

    In vitro layer separation of human dermal tissues via laser plasma-mediated ablation was successfully conducted for the first time. The ablation of wet dermis was generated using a 900 fs ultra-short pulsed laser with the wavelength centred at 1552 nm. The ablation and separation results were imaged and measured by scanning electron microscopy. The dermis ablation threshold was determined as 9.65 {+-} 1.21 J cm{sup -2} and the incubation factor was found as 0.46 {+-} 0.03. Histological examinations were performed to find proper laser parameters for dermis ablation and separation with minimal thermal damage. No thermal damage was found in the single line ablation results when the pulse overlap rate was not over 5 pulses {mu}m{sup -1}. Even in the multi-line ablation, thermal damage was insignificant and the lateral damage zone was generally within 5 {mu}m in the results with 100 continuously repeated line scans. The separation of a whole piece of wet dermal tissue into two thin layers was presented. Several separation tests with different layer thicknesses from 200 to 600 {mu}m were completed. The unevenness of the separated layers was generally under 10%. The cohesion and morphology of the separated tissue layers were not altered.

  3. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  4. Human Sarcoma growth is sensitive to small-molecule mediated AXIN stabilization.

    Directory of Open Access Journals (Sweden)

    Alessandra De Robertis

    Full Text Available Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas.

  5. EFFECT OF TITANIUM-ALLOY PARTICLES ON EXPRESSION OF MEDIATING-RELATED GENES IN HUMAN OSTEOBLASTS

    Institute of Scientific and Technical Information of China (English)

    杨旭; 杨庆铭; 邓廉夫; 许福平; 张玥

    2004-01-01

    Objective To study mRNA expression of the mediating-related genes in osteoblasts after exposure to Titanium-alloy particles and cytochalasin-D. Methods Human osteoblasts were isolated from trabecular fragments and exposed to 0. 01% (v/v) titanium-alloy particles for 8h. Pretreatment of osteoblasts with cytochalasinD( 5μmol/L ) to prevent phagocytosis prior to the addition of Titanium-alloy particles, mRNA expression of RANKL,OPG, and IL-6 was semi-quantified by RT-PCR. Results Expressions of RANKL, OPG, and lL-6 mRNA at respective levels were found in osteoblasts under normal condition, which did not show significant changes in the samples treated with cytochalasin-D but without particles. Exposure of osteoblasts to particles after being treated with or without cytochalasin-D altered mRNA expression of these genes, however, an increase in the ratio of RANKL/ OPG mRNA was only found in samples without cytochalassin-D treatment. Conclusion Changes of expression of RANKL, OPG, and IL-6 mRNA in osteoblasts after exposure to titanium-alloy particles were independent of phagocytosis, whereas phagocytosis has a direct effect on their ability to stimulate osteoclast differentiation and function.

  6. Feral Cat Globetrotters: genetic traces of historical human-mediated dispersal.

    Science.gov (United States)

    Koch, Katrin; Algar, Dave; Schwenk, Klaus

    2016-08-01

    Endemic species on islands are highly susceptible to local extinction, in particular if they are exposed to invasive species. Invasive predators, such as feral cats, have been introduced to islands around the world, causing major losses in local biodiversity. In order to control and manage invasive species successfully, information about source populations and level of gene flow is essential. Here, we investigate the origin of feral cats of Hawaiian and Australian islands to verify their European ancestry and a potential pattern of isolation by distance. We analyzed the genetic structure and diversity of feral cats from eleven islands as well as samples from Malaysia and Europe using mitochondrial DNA (ND5 and ND6 regions) and microsatellite DNA data. Our results suggest an overall European origin of Hawaiian cats with no pattern of isolation by distance between Australian, Malaysian, and Hawaiian populations. Instead, we found low levels of genetic differentiation between samples from Tasman Island, Lana'i, Kaho'olawe, Cocos (Keeling) Island, and Asia. As these populations are separated by up to 10,000 kilometers, we assume an extensive passive dispersal event along global maritime trade routes in the beginning of the 19th century, connecting Australian, Asian, and Hawaiian islands. Thus, islands populations, which are characterized by low levels of current gene flow, represent valuable sources of information on historical, human-mediated global dispersal patterns of feral cats.

  7. Human-mediated dispersal of seeds by the airflow of vehicles.

    Science.gov (United States)

    von der Lippe, Moritz; Bullock, James M; Kowarik, Ingo; Knopp, Tatjana; Wichmann, Matthias C; Wichmann, Matthias

    2013-01-01

    Human-mediated dispersal is known as an important driver of long-distance dispersal for plants but underlying mechanisms have rarely been assessed. Road corridors function as routes of secondary dispersal for many plant species but the extent to which vehicles support this process remains unclear. In this paper we quantify dispersal distances and seed deposition of plant species moved over the ground by the slipstream of passing cars. We exposed marked seeds of four species on a section of road and drove a car along the road at a speed of 48 km/h. By tracking seeds we quantified movement parallel as well as lateral to the road, resulting dispersal kernels, and the effect of repeated vehicle passes. Median distances travelled by seeds along the road were about eight meters for species with wind dispersal morphologies and one meter for species without such adaptations. Airflow created by the car lifted seeds and resulted in longitudinal dispersal. Single seeds reached our maximum measuring distance of 45 m and for some species exceeded distances under primary dispersal. Mathematical models were fit to dispersal kernels. The incremental effect of passing vehicles on longitudinal dispersal decreased with increasing number of passes as seeds accumulated at road verges. We conclude that dispersal by vehicle airflow facilitates seed movement along roads and accumulation of seeds in roadside habitats. Dispersal by vehicle airflow can aid the spread of plant species and thus has wide implications for roadside ecology, invasion biology and nature conservation.

  8. Myricitrin Inhibits Acrylamide-Mediated Cytotoxicity in Human Caco-2 Cells by Preventing Oxidative Stress

    Science.gov (United States)

    Chen, Wei; Feng, Lina; Shen, Yang; Su, Hongming; Li, Ya; Zhuang, Jingjing; Zhang, Lingxia; Zheng, Xiaodong

    2013-01-01

    Oxidative stress was thought to be associated with acrylamide cytotoxicity, but the link between oxidative stress and acrylamide cytotoxicity in the gastrointestinal tract, the primary organ in contact with dietary acrylamide, is still unclear. This study was conducted to evaluate the antioxidant activity of natural dietary compound myricitrin and its protective role against acrylamide cytotoxicity. We found that myricitrin can effectively scavenge multiple free radicals (including DPPH free radical, hydroxyl radical, and ABTS free radical) in a concentration-dependent manner. Our results further indicated that the presence of myricitrin (2.5–10 μg/mL) was found to significantly inhibit acrylamide-induced cytotoxicity in human gastrointestinal Caco-2 cells. Moreover, acrylamide-induced cytotoxicity is closely related to oxidative stress in Caco-2 cells. Interestingly, myricitrin was able to suppress acrylamide toxicity by inhibiting ROS generation. Taken together, these results demonstrate that myricitrin had a profound antioxidant effect and can protect against acrylamide-mediated cytotoxicity. PMID:24224177

  9. Land use mediates riverine nitrogen export under the dominant influence of human activities

    Science.gov (United States)

    Chen, Binhui; Chang, Scott X.; Lam, Shu Kee; Erisman, Jan Willem; Gu, Baojing

    2017-09-01

    Riverine nitrogen (N) export is a crucial process that links upstream and downstream ecosystems and coastal zones. However, the driving forces of riverine N export that is closely related to water N pollution are still not well understood. In this study, we used a mass balance approach to quantify the sources of N discharge and analyzed the effect of land use composition on riverine N export, taking Zhejiang Province, China as a case study. We found that the total reactive N discharge to rivers in Zhejiang increased from 0.22 to 0.26 Tg yr‑1 from 2000 to 2015. At the watershed scale, our estimate of N export agrees well with the monitored riverine N concentration in the eight major watersheds in Zhejiang. Direct discharge of domestic wastewater and effluents from wastewater treatment plants are dominant sources of riverine N export, followed by agricultural non-point sources. Although riverine N export increases with the increasing proportion of urban and agricultural land uses, we did not find any relationship between land use change and changes in riverine N export. This suggests that the dominant factor affecting riverine N export should be human activities (e.g. wastewater discharge and fertilization level), while land use only mediates riverine N export.

  10. Human-mediated dispersal of seeds by the airflow of vehicles.

    Directory of Open Access Journals (Sweden)

    Moritz von der Lippe

    Full Text Available Human-mediated dispersal is known as an important driver of long-distance dispersal for plants but underlying mechanisms have rarely been assessed. Road corridors function as routes of secondary dispersal for many plant species but the extent to which vehicles support this process remains unclear. In this paper we quantify dispersal distances and seed deposition of plant species moved over the ground by the slipstream of passing cars. We exposed marked seeds of four species on a section of road and drove a car along the road at a speed of 48 km/h. By tracking seeds we quantified movement parallel as well as lateral to the road, resulting dispersal kernels, and the effect of repeated vehicle passes. Median distances travelled by seeds along the road were about eight meters for species with wind dispersal morphologies and one meter for species without such adaptations. Airflow created by the car lifted seeds and resulted in longitudinal dispersal. Single seeds reached our maximum measuring distance of 45 m and for some species exceeded distances under primary dispersal. Mathematical models were fit to dispersal kernels. The incremental effect of passing vehicles on longitudinal dispersal decreased with increasing number of passes as seeds accumulated at road verges. We conclude that dispersal by vehicle airflow facilitates seed movement along roads and accumulation of seeds in roadside habitats. Dispersal by vehicle airflow can aid the spread of plant species and thus has wide implications for roadside ecology, invasion biology and nature conservation.

  11. Langhans giant cells from M. tuberculosis-induced human granulomas cannot mediate mycobacterial uptake.

    Science.gov (United States)

    Lay, G; Poquet, Y; Salek-Peyron, P; Puissegur, M-P; Botanch, C; Bon, H; Levillain, F; Duteyrat, J-L; Emile, J-F; Altare, F

    2007-01-01

    Tuberculosis is characterized by a tight interplay between Mycobacterium tuberculosis (M. tb) and host cells within granulomas. These cellular aggregates restrain M. tb spreading but do not kill all bacilli, which persist for years. A more detailed investigation of the interaction between M. tb and granuloma cells is needed to improve our understanding of this persistence and to explain the physiopathology of tuberculosis. In the present study, a recently developed in vitro human model of tuberculous granulomas has been used to analyse the modulation of granuloma cell differentiation by M. tb, in comparison to poorly virulent mycobacteria, which do not persist. It is reported that whilst all mycobacteria species induce granuloma formation, only M. tb triggers the differentiation of granuloma macrophages into very large multinucleated giant cells (MGCs) that are unable to mediate any bacterial uptake. This loss of function is not due to cell quiescence, as MGCs still display NADPH oxidase activity, but it correlates with decreased expression of phagocytosis receptors. This phenomenon is specific for the virulent species of M. tuberculosis complex, as poorly virulent species only induce the formation of small multinucleated cells (MCs) with conserved mycobacterial uptake ability, which never reach the MGC differentiation stage. The phenotype of MGCs thus strongly resembles mature dendritic cells with a loss of microbial uptake ability, despite conserved antigen presentation. In M. tb-induced granulomas, MGCs thus seem to be devoted to the destruction of bacilli that have been ingested in previous differentiation stages, ie in macrophages and MCs.

  12. microRNA-21 mediates stretch-induced osteogenic differentiation in human periodontal ligament stem cells.

    Science.gov (United States)

    Wei, Fulan; Liu, Dongxu; Feng, Cheng; Zhang, Fan; Yang, Shuangyan; Hu, Yijun; Ding, Gang; Wang, Songlin

    2015-02-01

    microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3'-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch.

  13. Adenovirus vector-mediated RNA interference for the inhibition of human parvovirus B19 replication.

    Science.gov (United States)

    Brandt, Marius R G; Kirste, Ariane G; Pozzuto, Tanja; Schubert, Steffen; Kandolf, Reinhard; Fechner, Henry; Bock, C-Thomas; Kurreck, Jens

    2013-09-01

    Human parvovirus B19 (B19V) has been considered to cause acute and chronic myocarditis, which is accompanied by endothelial dysfunction. Currently, no causative treatment option for B19V-infections is available. Since RNA interference (RNAi) has proven to be a highly potent antiviral approach, the aim of the current study was to develop an RNAi-based strategy to inhibit B19V replication. Three B19V-VP2-specific short hairpin RNAs (shRNAs) were designed and tested for their silencing activity in reporter assays and the expression cassette of the best one was introduced into an adenoviral shuttle vector (Ad5). B19V-permissive UT7/Epo-S1 cells were infected with B19V and the RNAi triggers were delivered by the adenoviral vector (Ad5shVP2) 24h thereafter. The shRNA targeting the B19V-VP2 gene significantly suppressed VP2 mRNA levels as determined by quantitative RT-PCR. Additionally, also the expression levels of the non-targeted non-structural B19V-NS1 mRNA were strongly reduced. Our results demonstrate that vector-mediated delivery of shRNA expression cassettes targeting the structural B19-VP2 gene is a suitable approach to inhibit B19V replication.

  14. Inositol Hexakisphosphate Mediates Apoptosis in Human Breast Adenocarcinoma MCF-7 Cell Line via Intrinsic Pathway

    Science.gov (United States)

    Agarwal, Rakhee; Ali, Nawab

    2010-04-01

    Inositol polyphosphates (InsPs) are naturally occurring compounds ubiquitously present in plants and animals. Inositol hexakisphosphate (InsP6) is the most abundant among all InsPs and constitutes the major portion of dietary fiber in most cereals, legumes and nuts. Certain derivatives of InsPs also regulate cellular signaling mechanisms. InsPs have also been shown to reduce tumor formation and induce apoptosis in cancerous cells. Therefore, in this study, the effects of InsPs on apoptosis were studied in an attempt to investigate their potential anti-cancer therapeutic application and understand their mechanism of action. Acridine orange and ethidium bromide staining suggested that InsP6 dose dependently induced apoptosis in human breast adenocarcinoma MCF-7 cells. Among InsPs tested (InsP3, InsP4, InsP5, and InsP6), InsP6 was found to be the most effective in inducing apoptosis. Furthermore, effects of InsP6 were found most potent inducing apoptosis. Etoposide, the drug known to induce apoptosis in both in vivo and in vitro, was used as a positive control. Western blotting experiments using specific antibodies against known apoptotic markers suggested that InsP6 induced apoptotic changes were mediated via an intrinsic apoptotic pathway.

  15. A Comparative Study of Early Afterdepolarization-Mediated Fibrillation in Two Mathematical Models for Human Ventricular Cells.

    Science.gov (United States)

    Zimik, Soling; Vandersickel, Nele; Nayak, Alok Ranjan; Panfilov, Alexander V; Pandit, Rahul

    2015-01-01

    Early afterdepolarizations (EADs), which are abnormal oscillations of the membrane potential at the plateau phase of an action potential, are implicated in the development of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1) a normal action potential (AP) with no EADs, (2) an AP with EADs, and (3) an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two- and three-dimensional domains, in the presence of EADs, we find the following wave types: (A) waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves); (B) waves driven only by the L-type calcium current (Ca-mediated waves); (C) phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves) in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca-mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model.

  16. A Comparative Study of Early Afterdepolarization-Mediated Fibrillation in Two Mathematical Models for Human Ventricular Cells.

    Directory of Open Access Journals (Sweden)

    Soling Zimik

    Full Text Available Early afterdepolarizations (EADs, which are abnormal oscillations of the membrane potential at the plateau phase of an action potential, are implicated in the development of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1 a normal action potential (AP with no EADs, (2 an AP with EADs, and (3 an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two- and three-dimensional domains, in the presence of EADs, we find the following wave types: (A waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves; (B waves driven only by the L-type calcium current (Ca-mediated waves; (C phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca-mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model.

  17. CD8+ Granzyme B+–Mediated Tissue Injury vs. CD4+IFNγ+–Mediated Parasite Killing in Human Cutaneous Leishmaniasis

    Science.gov (United States)

    Santos, Claire da Silva; Boaventura, Viviane; Ribeiro Cardoso, Cristina; Tavares, Natalia; Lordelo, Morgana J; Noronha, Almério; Costa, Jackson; Borges, Valéria M.; de Oliveira, Camila I; Van Weyenbergh, Johan; Barral, Aldina; Barral-Netto, Manoel; Brodskyn, Cláudia Ida

    2013-01-01

    A protective or deleterious role of CD8+T cells in human cutaneous leishmaniasis (CL) has been debated. The present report explores the participation of CD8+T cells in disease pathogenesis as well as in parasite killing. CD8+T cells accumulated in CL lesions as suggested by a higher frequency of CD8+CD45RO+T cells and CD8+CLA+T cells compared with peripheral blood mononuclear cells. Upon Leishmania braziliensis restimulation, most of the CD8+T cells from the lesion expressed cytolytic markers, CD107a and granzyme B. Granzyme B expression in CL lesions positively correlated with lesion size and percentage of TUNEL-positive cells. We also observed a significantly higher percentage of TUNEL-positive cells and granzyme B expression in the biopsies of patients showing a more intense necrotic process. Furthermore, coculture of infected macrophages and CD8+T lymphocytes resulted in the release of granzyme B, and the use of granzyme B inhibitor, as well as z-VAD, Fas:Fc, or anti-IFN-γ, had no effect upon parasite killing. However, coculture of infected macrophages with CD4+T cells strongly increased parasite killing, which was completely reversed by anti-IFN-γ. Our results reveal a dichotomy in human CL: CD8+ granzyme B+T cells mediate tissue injury, whereas CD4+IFN-γ+T cells mediate parasite killing. PMID:23321919

  18. Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin.

    Science.gov (United States)

    Afaq, Farrukh; Zaid, Mohammad Abu; Khan, Naghma; Dreher, Mark; Mukhtar, Hasan

    2009-06-01

    Solar ultraviolet (UV) radiation, particularly its UVB (290-320 nm) component, is the primary cause of many adverse biological effects including photoageing and skin cancer. UVB radiation causes DNA damage, protein oxidation and induces matrix metalloproteinases (MMPs). Photochemoprevention via the use of botanical antioxidants in affording protection to human skin against UVB damage is receiving increasing attention. Pomegranate, from the tree Punica granatum, contains anthocyanins and hydrolysable tannins and possesses strong antioxidant and anti-tumor-promoting properties. In this study, we determined the effect of pomegranate-derived products--POMx juice, POMx extract and pomegranate oil (POMo)--against UVB-mediated damage using reconstituted human skin (EpiDerm(TM) FT-200). EpiDerm was treated with POMx juice (1-2 microl/0.1 ml/well), POMx extract (5-10 microg/0.1 ml/well) and POMo (1-2 microl/0.1 ml/well) for 1 h prior to UVB (60 mJ/cm(2)) irradiation and was harvested 12 h post-UVB to assess protein oxidation, markers of DNA damage and photoageing by Western blot analysis and immunohistochemistry. Pretreatment of Epiderm with pomegranate-derived products resulted in inhibition of UVB-induced (i) cyclobutane pyrimidine dimers (CPD), (ii) 8-dihydro-2'-deoxyguanosine (8-OHdG), (iii) protein oxidation and (iv) proliferating cell nuclear antigen (PCNA) protein expression. We also found that pretreatment of Epiderm with pomegranate-derived products resulted in inhibition of UVB-induced (i) collagenase (MMP-1), (ii) gelatinase (MMP-2, MMP-9), (iii) stromelysin (MMP-3), (iv) marilysin (MMP-7), (v) elastase (MMP-12) and (vi) tropoelastin. Gelatin zymography revealed that pomegranate-derived products inhibited UVB-induced MMP-2 and MMP-9 activities. Pomegranate-derived products also caused a decrease in UVB-induced protein expression of c-Fos and phosphorylation of c-Jun. Collectively, these results suggest that all three pomegranate-derived products may be useful

  19. Prenatal Nicotinic Exposure Upregulates Pulmonary C-fiber NK1R Expression to Prolong Pulmonary C-fiber-Mediated Apneic Response

    Science.gov (United States)

    Zhao, Lei; Zhuang, Jianguo; Zang, Na; Lin, Yong; Lee, Lu-Yuan; Xu, Fadi

    2015-01-01

    Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChRs or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin. PMID:26524655

  20. Identification of key amino acid differences contributing to neonicotinoid sensitivity between two nAChR α subunits from Pardosa pseudoannulata.

    Science.gov (United States)

    Meng, Xiangkun; Zhang, Yixi; Guo, Beina; Sun, Huahua; Liu, Chuanjun; Liu, Zewen

    2015-01-01

    Chemical insecticides are still primary methods to control rice planthoppers in China, which not only cause environmental pollution, insecticide residue and insecticide resistance, but also have negative effects on natural enemies, such as Pardosa pseudoannulata (the pond wolf spider), an important predatory enemy of rice planthoppers. Neonicotinoids insecticides, such as imidacloprid and thiacloprid, are insect-selective nAChRs agonists that are used extensively in the areas of crop protection and animal health, but have hypotoxicity to P. pseudoannulata. In the present study, two nAChR α subunits, Ppα1 or Ppα8, were found to be successfully expressed with rβ2 in Xenopus oocytes, but with much different sensitivity to imidacloprid and thiacloprid on two recombinant receptors Ppα1/rβ2 and Ppα8/rβ2. Key amino acid differences were found in and between the important loops for ligand binding. In order to well understand the relationship between the amino acid differences and neonicotinoid sensitivities, different segments in Ppα8 or Ppα1 with key amino acid differences were introduced into the corresponding regions of Ppα1 or Ppα8 to construct chimeras and then co-expressed with rβ2 subunit in Xenopus oocytes. The results from chimeras of both Ppα8 and Ppα1 showed that segments Δ5, Δ6, and Δ7 contributed to neonicotinoid sensitivities directly between two receptors. Although the segment Δ4 including all loop B region had no direct influences on neonicotinoid sensitivities, it could more remarkably influence neonicotinoid sensitivities when co-introductions with Δ5, Δ6 or Δ7. So, key amino acid differences in these four segments were important to neonicotinoid sensitivities, but the difference in Δ4 was likely ignored because of its indirect effects.

  1. Nicotine analogues as potential therapeutic agents in Parkinson’s disease by targeting nicotinic acetylcholine receptors (nAChRs in astrocytes

    Directory of Open Access Journals (Sweden)

    Valentina Echeverria Moran

    2015-02-01

    Full Text Available Parkinson’s disease (PD is a relatively common disorder of the Central Nervous System (CNS, whose etiology is characterized by a selective and progressive degeneration of dopaminergic neurons, and the presence of Lewy bodies in the pars compacta of the substantia nigra, thus dopamine depletion in the striatum. Patients with this disease suffer from tremors, slowness of movements, gait instability, rigidity, and may also present functional disability, reduced quality of life, and rapid cognitive decline. The prevalence of this disease is in a range of 107-187 per 100,000 inhabitants. Previous studies have shown that nicotine exerts beneficial effects in patients with PD and in in vitro and in vivo models of this disease. Astrocytes have an important role in the immune system, and that nicotine might be able to reduce inflammation-induced activation of pro-apoptotic signaling in PD. Nicotine might exert its effect through activation of α7 nicotinic acetylcholine receptors (α7-nAChRs expressed in glial cells. Moreover, nicotine administration can protect dopaminergic neurons against degeneration by inhibiting astrocytes activation in the substantia nigra pars compacta (SNpc and therefore reducing inflammation. Besides this beneficial effect of nicotine, its continuing use can induce toxicity and cause dependency. To counteract this effect, nicotine analogues have risen as an important therapeutic approach to maintain nicotine´s beneficial effects, but avoid its toxicity. Since astrocytes might drive chronic inflammatory processes in PD, therefore increasing neuronal vulnerability to damage, the administration of nicotine analogues in astrocytes is of interest to diminish neuronal death. In this work, we assess the role of different nicotine analogues in astrocytes following rotenone stimuli, and determine whether the possible beneficial effects of nicotine are via activation of α7-nAChRs.

  2. The human body on Exhibit: promoting socio-cultural mediations in a science museum

    OpenAIRE

    Silvania Sousa do Nascimento

    2008-01-01

    This paper discusses three mediation concept approaches and, consequently, three facets of mediator action. The approaches presented start with a bibliographical review of the concept of mediation present in education and scientific communication studies. These approaches serve as a basis for interpreting a semi-directive interview with the director of the Museum of Morphological Sciences of the Federal University of Minas Gerais (UFMG). They also help us reflect on the complexity of organ...

  3. Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

    Directory of Open Access Journals (Sweden)

    Ami Oizumi

    Full Text Available Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD. A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes", which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii S1P induces the production

  4. Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Kleve MG

    2011-07-01

    Full Text Available Md. Zakir Hossain1, Maurice G Kleve21Applied Biosciences (Bionanotechnology Research, Department of Applied Science, 2Molecular Biotechnology and Microscopy Laboratory, Department of Biology, University of Arkansas at Little Rock, Little Rock, Arkansas, USABackground: The ability to evade apoptosis is one of the key properties of cancer. The apoptogenic effect of nickel nanowires (Ni NWs on cancer cell lines has never been adequately addressed. Due to the unique physicochemical characteristics of Ni NWs, we envision the development of a novel anticancer therapeutics specifically for pancreatic cancer. Thus, we investigated whether Ni NWs induce ROS-mediated apoptosis in human pancreatic adenocarcinoma (Panc-1 cells. Methods: In this study Ni NWs were fabricated using the electrodeposition method. Synthesized Ni NWs were physically characterized by energy dispersive X-ray analysis, UV-Vis spectroscopy of NanoDrop 2000 (UV-Vis, magnetization study, scanning electron microscopy, and transmission electron microscopy. Assessment of morphological apoptotic characteristics by phase contrast microscopy (PCM, Ni-NWs-induced apoptosis staining with ethidium bromide (EB and acridine orange (AO followed by fluorescence microscopy (FM was performed. For molecular biological and biochemical characterization, Panc-1 cell culture and cytotoxic effect of Ni NWs were determined by using 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT assay. Quantitative apoptosis was analyzed by flow cytometry staining with propidium iodide through cell cycle arrest and generation of ROS using 2', 7'-dichlorofluorescein diacetate fluorescence intensity. In all experiments, Panc-1 cancer cells without any treatment were used as the negative controls.Results: The intracellular uptake of Ni NWs through endocytosis by Panc-1 cells was observed by PCM. EB and AO staining of FM and MTT assay qualitatively and quantitatively confirmed the extent of apoptosis. Flow

  5. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Xianglian Ge

    2016-01-01

    Full Text Available Clustered interspaced short palindromic repeats (CRISPR/CRISPR-associated protein 9 (Cas9-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

  6. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells.

    Science.gov (United States)

    Ge, Xianglian; Xi, Haitao; Yang, Fayu; Zhi, Xiao; Fu, Yanghua; Chen, Ding; Xu, Ren-He; Lin, Ge; Qu, Jia; Zhao, Junzhao; Gu, Feng

    2016-11-29

    Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

  7. Trichloroethylene-mediated cytotoxicity in human epidermal keratinocytes is mediated by the rapid accumulation of intracellular calcium: Interception by naringenin.

    Science.gov (United States)

    Ali, F; Khan, A Q; Khan, R; Sultana, S

    2016-02-01

    Industrial solvents pose a significant threat to the humankind. The mechanisms of their toxicity still remain in debate. Trichloroethylene (TCE) is a widespread industrial solvent responsible for severe liver dysfunction, cutaneous toxicity in occupationally exposed humans. We utilized an in vitro system of human epidermal keratinocyte (HaCaT) cells in this study to avoid complex cell and extracellular interactions. We report the cytotoxicity of organic solvent TCE in HaCaT and its reversal by a natural flavanone, naringenin (Nar). The cytotoxicity was attributed to the rapid intracellular free calcium (Ca(2+)) release, which might lead to the elevation of protein kinase C along with robust free radical generation, instability due to energy depletion, and sensitization of intracellular stress signal transducer nuclear factor κB. These effects were actually seen to induce significant amount of genomic DNA fragmentation. Furthermore, all these effects of TCE were effectively reversed by the treatment of Nar, a natural flavanone. Our studies identify intracellular Ca as a unique target used by organic solvents in the cytotoxicity and highlight the Ca(2+) ion stabilizer properties of Nar.

  8. Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Weiming; Gorman, P.A.; Rider, S.H.; Hedge, P.J.; Moore, G.; Prichard, C.; Sheer, D.; Solomon, E. (Imperial Cancer Research Fund, London (England))

    1988-11-01

    The authors used somatic-cell hybrids, containing as their only human genetic contribution part or all of chromosome 17, as donors for chromosome-mediated gene transfer. A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for >20 loci located on chromosome 17. By combining the data from this chromosome-mediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization they propose the following order for these loci; pter-(TP53-RNP2-D17S1)-(MYH2-MYH1)-D17Z1-CRYB1-(ERBA1-GCSF-NGL)-acute promyelocytic leukemia breakpoint-RNU2-HOX2-(NGFR-COLIAI-MPO)-GAA-UMPH-GHC-TK1-GALK-qter. Using chromosome-mediated gene transfer, they have also regionally localized the random probes D17S6 to D17S19 on chromosome 17.

  9. Drosophila caliban, a nuclear export mediator, can function as a tumor suppressor in human lung cancer cells.

    Science.gov (United States)

    Bi, Xiaolin; Jones, Tamara; Abbasi, Fatima; Lee, Heuijung; Stultz, Brian; Hursh, Deborah A; Mortin, Mark A

    2005-12-15

    We previously showed that the Drosophila DNA binding homeodomain of Prospero included a 28 amino-acid sequence (HDA) that functions as a nuclear export signal. We describe here the identification of a protein we named Caliban, which can directly interact with the HDA. Caliban is homologous to human Sdccag1, which has been implicated in colon and lung cancer. Here we show that Caliban and Sdccag1 are mediators of nuclear export in fly and human cells, as interference RNA abrogates export of EYFP-HDA in normal fly and human lung cells. Caliban functions as a bipartite mediator nuclear export as the carboxy terminus binds HDA and the amino terminus itself functions as an NES, which directly binds the NES receptor Exportin. Finally, while non-cancerous lung cells have functional Sdccag1, five human lung carcinoma cell lines do not, even though Exportin still functions in these cells. Expression of fly Caliban in these human lung cancer cells restores EYFP-HDA nuclear export, reduces a cell's ability to form colonies on soft agar and reduces cell invasiveness. We suggest that Sdccag1 inactivation contributes to the transformed state of human lung cancer cells and that Caliban should be considered a candidate for use in lung cancer gene therapy.

  10. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes

    DEFF Research Database (Denmark)

    Lykke-Andersen, Søren; Chen, Yun; Ardal, Britt;

    2014-01-01

    Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogen......Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities...... yield alternative transcript isoforms that allow for differential expression of individual coencoded snoRNAs. Based on our findings, we hypothesize that snoRNA host genes need to be highly transcribed to accommodate high levels of snoRNA production and that the expression of individual sno...

  11. Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

    Directory of Open Access Journals (Sweden)

    Jessica Mühlhaus

    2014-11-01

    Full Text Available The thyroid hormone derivative 3-iodothyronamine (3-T1AM exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1 agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8. By RT-qPCR and locked-nucleic-acid (LNA in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs.

  12. Quantitative study on La3+ influx mediated by sodium-calcium exchanger in human lymphocytes

    Institute of Scientific and Technical Information of China (English)

    魏春英; 杨频

    2002-01-01

    Whether La3+ can enter human peripheral blood lymphocytes by the Na+/Ca2+ exchanger or not and the effect of La3+ on the Na+/Ca2+ exchanger activity are examined by fura-2 technique. And that whether La3+ is sequestered by intracellular organelles (mainly endoplasmic reticulum and mitochondria) is studied by this method. La3+ uptake is obviously stimulated by pretreating the cells with ouabain and by removing extracellular Na+, and intracellular La3+ concentration ([La3+]i) is directly proportional to its extracellular concentration ([La3+]o). But when [La3+]o exceeds 0.4 mmol/L, the 340/380 nm ratio of fluorescence is no longer varied and the maximum [La3+]i is 1.5×10-12 mol@L-1. The higher concentration of La3+ (0.1 mmol/L) increases Na+/Ca2+ exchange-mediated calcium influx, but lower concentration (10 mmol/L) appears to block calcium influx. The results also suggest that cytosolic La3+ is transported by the ATP-dependent Ca2+ pump. Intracellular Ca2+ stores are depleted by ionomycin, and then ionomycin is added again during the period of La3+ uptake, the 340/380 nm ratio of fluorescence is also increased, these results indicate that La3+ is sequestered by intracellular organelles. A characterization of fura-2-La3+ interaction in solution simulating intracellular ionic composition (pH 7.05) shows that La3+ forms a 1:1 fura-2-La3+complex, and the apparent dissociation constant of La3+ for fura-2 (Kd) is 1.7×10-12 mol@L-1. In addition, the limit of detection of fura-2 for La3+ and Ca2+ is 10?12 and 10?8 mol@L-1 respectively.

  13. Fetal development of the elastic-fiber-mediated enthesis in the human middle ear.

    Science.gov (United States)

    Takanashi, Yoshitaka; Shibata, Shunichi; Katori, Yukio; Murakami, Gen; Abe, Shinichi; Rodríguez-Vázquez, Jose Francisco; Kawase, Tetsuaki

    2013-10-01

    In the human middle ear, the annular ligament of the incudostapedial joint and the insertions of the tensor tympani and stapedius muscles contain abundant elastic fibers; i.e., the elastic-fiber-mediated entheses. Hyaluronan also coexists with the elastic fibers. In the present study using immunohistochemistry, we demonstrated the distribution of elastin not only in the incudostapedial joint but also in the other two joints of the middle ear in adults and fetuses. In adults, the expression of elastin did not extend out of the annular ligament composed of mature elastic fibers but clearly overlapped with it. Electron microscopic observations of the annular ligament demonstrated a few microfibrils along the elastic fibers. Thus, in contrast to the vocal cord, the middle ear entheses seemed not to contain elaunin and oxytalan fibers. In mid-term fetuses (at approximately 15-16 weeks of gestation) before opening of the external acoustic meatus, the incudostapedial joint showed abundant elastic fibers, but the incudomalleolar and stapediovestibular joints did not. At this stage, hyaluronan was not colocalized, but distributed diffusely in loose mesenchymal tissues surrounding the ear ossicles. Therefore, fetal development of elastin and elastic fibers in the middle ear entheses is unlikely to require acoustic oscillation. In late-stage fetuses (25-30 weeks), whose ear ossicles were almost the same size as those in adults, we observed bundling and branching of elastic fibers. However, hyaluronan expression was not as strong as in adults. Colocalization between elastic fibers and hyaluronan appeared to be a result of postnatal maturation of the entheses.

  14. Modified Titanium Surface-Mediated Effects on Human Bone Marrow Stromal Cell Response

    Directory of Open Access Journals (Sweden)

    Amol Chaudhari

    2013-11-01

    Full Text Available Surface modification of titanium implants is used to enhance osseointegration. The study objective was to evaluate five modified titanium surfaces in terms of cytocompatibility and pro-osteogenic/pro-angiogenic properties for human mesenchymal stromal cells: amorphous microporous silica (AMS, bone morphogenetic protein-2 immobilized on AMS (AMS + BMP, bio-active glass (BAG and two titanium coatings with different porosity (T1; T2. Four surfaces served as controls: uncoated Ti (Ti, Ti functionalized with BMP-2 (Ti + BMP, Ti surface with a thickened titanium oxide layer (TiO2 and a tissue culture polystyrene surface (TCPS. The proliferation of eGFP-fLuc (enhanced green fluorescence protein-firefly luciferase transfected cells was tracked non-invasively by fluorescence microscopy and bio-luminescence imaging. The implant surface-mediated effects on cell differentiation potential was tracked by determination of osteogenic and angiogenic parameters [alkaline phosphatase (ALP; osteocalcin (OC; osteoprotegerin (OPG; vascular endothelial growth factor-A (VEGF-A]. Unrestrained cell proliferation was observed on (unfunctionalized Ti and AMS surfaces, whereas BAG and porous titanium coatings T1 and T2 did not support cell proliferation. An important pro-osteogenic and pro-angiogenic potential of the AMS + BMP surface was observed. In contrast, coating the Ti surface with BMP did not affect the osteogenic differentiation of the progenitor cells. A significantly slower BMP-2 release from AMS compared to Ti supports these findings. In the unfunctionalized state, Ti was found to be superior to AMS in terms of OPG and VEGF-A production. AMS is suggested to be a promising implant coating material for bioactive agents delivery.

  15. Modified Titanium Surface-Mediated Effects on Human Bone Marrow Stromal Cell Response.

    Science.gov (United States)

    Chaudhari, Amol; Duyck, Joke; Braem, Annabel; Vleugels, Jozef; Petite, Hervé; Logeart-Avramoglou, Delphine; Naert, Ignace; Martens, Johan A; Vandamme, Katleen

    2013-11-28

    Surface modification of titanium implants is used to enhance osseointegration. The study objective was to evaluate five modified titanium surfaces in terms of cytocompatibility and pro-osteogenic/pro-angiogenic properties for human mesenchymal stromal cells: amorphous microporous silica (AMS), bone morphogenetic protein-2 immobilized on AMS (AMS + BMP), bio-active glass (BAG) and two titanium coatings with different porosity (T1; T2). Four surfaces served as controls: uncoated Ti (Ti), Ti functionalized with BMP-2 (Ti + BMP), Ti surface with a thickened titanium oxide layer (TiO₂) and a tissue culture polystyrene surface (TCPS). The proliferation of eGFP-fLuc (enhanced green fluorescence protein-firefly luciferase) transfected cells was tracked non-invasively by fluorescence microscopy and bio-luminescence imaging. The implant surface-mediated effects on cell differentiation potential was tracked by determination of osteogenic and angiogenic parameters [alkaline phosphatase (ALP); osteocalcin (OC); osteoprotegerin (OPG); vascular endothelial growth factor-A (VEGF-A)]. Unrestrained cell proliferation was observed on (un)functionalized Ti and AMS surfaces, whereas BAG and porous titanium coatings T1 and T2 did not support cell proliferation. An important pro-osteogenic and pro-angiogenic potential of the AMS + BMP surface was observed. In contrast, coating the Ti surface with BMP did not affect the osteogenic differentiation of the progenitor cells. A significantly slower BMP-2 release from AMS compared to Ti supports these findings. In the unfunctionalized state, Ti was found to be superior to AMS in terms of OPG and VEGF-A production. AMS is suggested to be a promising implant coating material for bioactive agents delivery.

  16. Exercise-mediated vasodilation in human obesity and metabolic syndrome: effect of acute ascorbic acid infusion

    Science.gov (United States)

    Limberg, Jacqueline K.; Kellawan, J. Mikhail; Harrell, John W.; Johansson, Rebecca E.; Eldridge, Marlowe W.; Proctor, Lester T.; Sebranek, Joshua J.

    2014-01-01

    We tested the hypothesis that infusion of ascorbic acid (AA), a potent antioxidant, would alter vasodilator responses to exercise in human obesity and metabolic syndrome (MetSyn). Forearm blood flow (FBF, Doppler ultrasound) was measured in lean, obese, and MetSyn adults (n = 39, 32 ± 2 yr). A brachial artery catheter was inserted for blood pressure monitoring and local infusion of AA. FBF was measured during dynamic handgrip exercise (15% maximal effort) with and without AA infusion. To account for group differences in blood pressure and forearm size, and to assess vasodilation, forearm vascular conductance (FVC = FBF/mean arterial blood pressure/lean forearm mass) was calculated. We examined the time to achieve steady-state FVC (mean response time, MRT) and the rise in FVC from rest to steady-state exercise (Δ, exercise − rest) before and during acute AA infusion. The MRT (P = 0.26) and steady-state vasodilator responses to exercise (ΔFVC, P = 0.31) were not different between groups. Intra-arterial infusion of AA resulted in a significant increase in plasma total antioxidant capacity (174 ± 37%). AA infusion did not alter MRT or steady-state FVC in any group (P = 0.90 and P = 0.85, respectively). Interestingly, higher levels of C-reactive protein predicted longer MRT (r = 0.52, P < 0.01) and a greater reduction in MRT with AA infusion (r = −0.43, P = 0.02). We concluded that AA infusion during moderate-intensity, rhythmic forearm exercise does not alter the time course or magnitude of exercise-mediated vasodilation in groups of young lean, obese, or MetSyn adults. However, systemic inflammation may limit the MRT to exercise, which can be improved with AA. PMID:25038148

  17. Signaling mechanisms mediated by G-protein coupled receptors in human platelets

    Institute of Scientific and Technical Information of China (English)

    Sheikh Arshad SAEED; Huma RASHEED; Faisal A Wahed FECTO; Mohammad Ilyas ACHAKZAI; Rahmat ALI; John Dennis CONNOR; Anwar-ul-Hassan GILANI

    2004-01-01

    AIM: The present study deals with the investigation of mechanisms involved in the synergistic interaction between epinephrine and arachidonic acid (AA). METHODS: Venous blood was taken from healthy human volunteers reported to be free of medications for one week. Platelet aggregation was monitored at 37 ℃ using Dual-channel Lumi-aggregometer. The resulting aggregation was recorded for 5 min by the measurement of light transmission as a function of time. RESULTS: The data show that a synergism in platelet aggregation mediated by subthreshold concentrations of epinephrine (1μmol/L) and AA (0.2μmol/L) was inhibited by the α2-receptor antagonist (yohimbine, IC50=0.6 μmol/L) and an inhibitor of AA-cyclooxygenase (COX), indomethacin (IC50=0.25 μmol/L).In examining receptor influence on intraplatelet signalling pathways, it was found that the synergistic effect was inhibited by calcium channel blockers, verapamil (IC50=0.4 μtmol/L) and diltiazem (IC50=2.5 μmol/L), as well as by low concentrations of inhibitors of phospholipase C (PLC) (U73122; IC50=0.2 μmol/L) and mitogens activated protein kinase (MAPK) (PD 98059; IC50=3.8 μmol/L). Herbimycin A, a specific inhibitor of tyrosine light chain kinase (TLCK), showed inhibition at IC50 value of 15 μmol/L, whereas chelerythrine, a protein kinase C (PKC)inhibitor, had no effect up to 20 μmol/L. CONCLUSION: These data suggest that synergism between epinephrine and AA in platelet aggregation is triggered through receptors coupled to G-protein, which in turn, activate PLC,COX, and MAP kinase-signaling pathways.

  18. Mediation of calcium oxalate crystal growth on human kidney epithelial cells with different degrees of injury

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Shen [Graduate School of Southern Medical University, Guangzhou 510515 (China); Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632 (China); Su Zexuan, E-mail: suz2008@126.com [The First Affiliated Hospital, Jinan University, Guangzhou 510632 (China); Yao Xiuqiong; Peng Hua; Deng Suiping [Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632 (China); Ouyang Jianming, E-mail: toyjm@jnu.edu.cn [Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632 (China)

    2012-05-01

    The current study examined the role of injured human kidney tubular epithelial cell (HKC) in the mediation of formation of calcium oxalate (CaOxa) crystals by means of scanning electronic microscopy and X-ray diffraction. HKC was injured using different concentrations of H{sub 2}O{sub 2}. Cell injury resulted in a significant decrease in cell viability and superoxide dismutase (SOD) concentration and an increase in the level of malondialdehyde (MDA) and expression of osteopontin (OPN). Injured cells not only promote nucleation and aggregation of CaOxa crystals, but also induce the formation of calcium oxalate monohydrate (COM) crystals that strongly adhere to cells. These results imply that injured HKCs promote stone formation by providing more nucleating sites for crystals, promoting the aggregation of crystals, and inducing the formation of COM crystals. - Graphical abstract: Injured cells promote nucleation and aggregation of CaOxa crystals, induce the formation of calcium oxalate monohydrate (COM) crystals. Highlights: Black-Right-Pointing-Pointer A direct nucleation and growth of CaOxa crystals on both normal and injured cells. Black-Right-Pointing-Pointer Stronger green fluorescence, i.e. OPN expression, was seen on the injury cell surface Black-Right-Pointing-Pointer Injured cells promote nucleation and aggregation of CaOxa crystals. Black-Right-Pointing-Pointer Injured cells induce the formation of calcium oxalate monohydrate crystals. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} decrease cell viability in a dose-dependent manner at 0.1-1 mmol/L.

  19. Heritability in the efficiency of nonsense-mediated mRNA decay in humans

    KAUST Repository

    Seoighe, Cathal

    2010-07-21

    Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. Conclusions: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3. © 2010 Seoighe, Gehring.

  20. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    LENUS (Irish Health Repository)

    Seoighe, Cathal

    2010-01-01

    BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  1. Distinct parietal sites mediate the influences of mood, arousal, and their interaction on human recognition memory.

    Science.gov (United States)

    Greene, Ciara M; Flannery, Oliver; Soto, David

    2014-12-01

    The two dimensions of emotion, mood valence and arousal, have independent effects on recognition memory. At present, however, it is not clear how those effects are reflected in the human brain. Previous research in this area has generally dealt with memory for emotionally valenced or arousing stimuli, but the manner in which interacting mood and arousal states modulate responses in memory substrates remains poorly understood. We investigated memory for emotionally neutral items while independently manipulating mood valence and arousal state by means of music exposure. Four emotional conditions were created: positive mood/high arousal, positive mood/low arousal, negative mood/high arousal, and negative mood/low arousal. We observed distinct effects of mood valence and arousal in parietal substrates of recognition memory. Positive mood increased activity in ventral posterior parietal cortex (PPC) and orbitofrontal cortex, whereas arousal condition modulated activity in dorsal PPC and the posterior cingulate. An interaction between valence and arousal was observed in left ventral PPC, notably in a parietal area distinct from the those identified for the main effects, with a stronger effect of mood on recognition memory responses here under conditions of relative high versus low arousal. We interpreted the PPC activations in terms of the attention-to-memory hypothesis: Increased arousal may lead to increased top-down control of memory, and hence dorsal PPC activation, whereas positive mood valence may result in increased activity in ventral PPC regions associated with bottom-up attention to memory. These findings indicate that distinct parietal sites mediate the influences of mood, arousal, and their interplay during recognition memory.

  2. Exercise-mediated vasodilation in human obesity and metabolic syndrome: effect of acute ascorbic acid infusion.

    Science.gov (United States)

    Limberg, Jacqueline K; Kellawan, J Mikhail; Harrell, John W; Johansson, Rebecca E; Eldridge, Marlowe W; Proctor, Lester T; Sebranek, Joshua J; Schrage, William G

    2014-09-15

    We tested the hypothesis that infusion of ascorbic acid (AA), a potent antioxidant, would alter vasodilator responses to exercise in human obesity and metabolic syndrome (MetSyn). Forearm blood flow (FBF, Doppler ultrasound) was measured in lean, obese, and MetSyn adults (n = 39, 32 ± 2 yr). A brachial artery catheter was inserted for blood pressure monitoring and local infusion of AA. FBF was measured during dynamic handgrip exercise (15% maximal effort) with and without AA infusion. To account for group differences in blood pressure and forearm size, and to assess vasodilation, forearm vascular conductance (FVC = FBF/mean arterial blood pressure/lean forearm mass) was calculated. We examined the time to achieve steady-state FVC (mean response time, MRT) and the rise in FVC from rest to steady-state exercise (Δ, exercise - rest) before and during acute AA infusion. The MRT (P = 0.26) and steady-state vasodilator responses to exercise (ΔFVC, P = 0.31) were not different between groups. Intra-arterial infusion of AA resulted in a significant increase in plasma total antioxidant capacity (174 ± 37%). AA infusion did not alter MRT or steady-state FVC in any group (P = 0.90 and P = 0.85, respectively). Interestingly, higher levels of C-reactive protein predicted longer MRT (r = 0.52, P exercise does not alter the time course or magnitude of exercise-mediated vasodilation in groups of young lean, obese, or MetSyn adults. However, systemic inflammation may limit the MRT to exercise, which can be improved with AA. Copyright © 2014 the American Physiological Society.

  3. MicroRNA Mediated Chemokine Responses in Human Airway Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Mythili Dileepan

    Full Text Available Airway smooth muscle (ASM cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.

  4. Luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

    Directory of Open Access Journals (Sweden)

    Poyil Pratheeshkumar

    Full Text Available Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

  5. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    Directory of Open Access Journals (Sweden)

    Cathal Seoighe

    Full Text Available BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  6. Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Lin, Junxiao; Ma, Biao; Fang, Jiehong; Wang, Ye; He, Haizhen; Lin, Wei; Su, Wei; Zhang, Mingzhou

    2017-03-01

    Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.

  7. The human body on Exhibit: promoting socio-cultural mediations in a science museum

    Directory of Open Access Journals (Sweden)

    Silvania Sousa do Nascimento

    2008-12-01

    Full Text Available This paper discusses three mediation concept approaches and, consequently, three facets of mediator action. The approaches presented start with a bibliographical review of the concept of mediation present in education and scientific communication studies. These approaches serve as a basis for interpreting a semi-directive interview with the director of the Museum of Morphological Sciences of the Federal University of Minas Gerais (UFMG. They also help us reflect on the complexity of organizing the objectives of a museum action that takes into account the transformational role of the meaning of objects in interaction with different socio-cultural subjects. In conclusion, the museum's purpose in organizing a museum action using socio-cultural mediation approach and with the mediator as a passeur libre among exhibit objects and visitors is highlighted.

  8. Mediated exodus of L-dopa from human epidermal Langerhans cells.

    Science.gov (United States)

    Falck, B; Bendsoe, N; Ronquist, G

    2004-03-01

    L-3,4-dihydroxyphenylalanine (L-dopa) is not metabolized within human epidermal Langerhans cells (LC); yet they can take up substantial amounts of this amino acid which subsequently can be released into the extracellular space. We recently reported that human epidermal energy metabolism is predominantly anaerobic and that the influx mechanism is a unidirectional L-dopa/proton counter-transport system and now we describe conditions for the mediated transport of L-dopa out of the LC. It is demonstrated that certain amino acids and one dipeptide can effectively trigger the efflux of L-dopa taken up by the LC.Thus, alpha-methyl-dopa (alpha-m-dopa), D-dopa and the dipeptide, met-ala at the outside of the plasma membrane stimulated the efflux of L-dopa from L-dopa loaded LC. Similar effects were achieved by a variety of other amino acids in the extracellular fluid while some other amino acids were inactive. The time required for 50% D-methionine-induced exodus of L-dopa from L-dopa loaded LC was in the range of 5-7 min and a complete exodus of L-dopa was attained at about 20 min of incubation. This dislocation of L-dopa to the extracellular fluid is interpreted as an expression of trans-stimulation. In the case of alpha-m-dopa, D-dopa and met-ala, which admittedly were not able to penetrate the plasma membrane of LC, the concept of trans-stimulation was given a new purport, since none of them were able to participate in an exchange reaction. Finally, it could be concluded that L-dopa escaped by a route different from the one responsible for L-dopa uptake in LC.Thus, while the influx of L-dopa supports extrusion of protons deriving from anaerobic glycolysis in the LC, L-dopa efflux can provide the cells with useful amino acids in an energy-saving way, altogether a remarkable biological process. From this follows that L-dopa has a biological function of its own, besides being a precursor in the catecholamine and pigment syntheses.

  9. Elimination of Dog-Mediated Human Rabies Deaths by 2030: Needs Assessment and Alternatives for Progress Based on Dog Vaccination

    OpenAIRE

    Ryan M. Wallace; Undurraga, Eduardo A; Blanton, Jesse D.; Cleaton, Julie; Franka, Richard

    2017-01-01

    Background Rabies imposes a substantial burden to about half of the world population. The World Health Organization (WHO), World Organization for Animal Health, and the Food and Agriculture Organization have set the goal of eliminating dog-mediated human rabies deaths by 2030. This could be achieved largely by massive administration of post-exposure prophylaxis—in perpetuity—, through elimination of dog rabies, or combining both. Here, we focused on the resources needed for the elimination...

  10. Immunity and AAV-mediated gene therapy for muscular dystrophies in large animal models and human trials

    OpenAIRE

    2011-01-01

    Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery ...

  11. Voyage of discovery? A comment on Koch et al. "A voyage to Terra Australis: human-mediated dispersal of cats".

    Science.gov (United States)

    Andrew, Rose L; Smith, Deane; Gorrell, Jamieson C; Janes, Jasmine K

    2016-12-07

    The origins of feral cats in Australia may be understood with the help of molecular studies, but it is important that hypotheses be tested with appropriate sampling and methodology. We point out several shortcomings in the analysis by Koch et al. (BMC Evol Biol 15:262, 2015; A voyage to Terra Australis: human-mediated dispersal of cats. Dryad Digital Repository, 2015), present a reanalysis of part of the study and discuss the challenges of elucidating the early history of feral cats.

  12. Elimination of Dog-Mediated Human Rabies Deaths by 2030: Needs Assessment and Alternatives for Progress Based on Dog Vaccination

    OpenAIRE

    Wallace, Ryan M.; Eduardo A Undurraga; Jesse D. Blanton; Cleaton, Julie; Franka, Richard

    2017-01-01

    Background Rabies imposes a substantial burden to about half of the world population. The World Health Organization (WHO), World Organization for Animal Health, and the Food and Agriculture Organization have set the goal of eliminating dog-mediated human rabies deaths by 2030. This could be achieved largely by massive administration of post-exposure prophylaxis—in perpetuity—, through elimination of dog rabies, or combining both. Here, we focused on the resources needed for the elimination of...

  13. Human SLURP-1 and SLURP-2 Proteins Acting on Nicotinic Acetylcholine Receptors Reduce Proliferation of Human Colorectal Adenocarcinoma HT-29 Cells.

    Science.gov (United States)

    Lyukmanova, E N; Shulepko, M A; Bychkov, M L; Shenkarev, Z O; Paramonov, A S; Chugunov, A O; Arseniev, A S; Dolgikh, D A; Kirpichnikov, M P

    2014-10-01

    Human secreted Ly-6/uPAR related proteins (SLURP-1 and SLURP-2) are produced by various cells, including the epithelium and immune system. These proteins act as autocrine/paracrine hormones regulating the growth and differentiation of keratinocytes and are also involved in the control of inflammation and malignant cell transformation. These effects are assumed to be mediated by the interactions of SLURP-1 and SLURP-2 with the α7 and α3β2 subtypes of nicotinic acetylcholine receptors (nAChRs), respectively. Available knowledge about the molecular mechanism underling the SLURP-1 and SLURP-2 effects is very limited. SLURP-2 remains one of the most poorly studied proteins of the Ly-6/uPAR family. In this study, we designed for the first time a bacterial system for SLURP-2 expression and a protocol for refolding of the protein from cytoplasmic inclusion bodies. Milligram quantities of recombinant SLURP-2 and its 13C-15N-labeled analog were obtained. The recombinant protein was characterized by NMR spectroscopy, and a structural model was developed. A comparative study of the SLURP-1 and SLURP-2 effects on the epithelial cell growth was conducted using human colorectal adenocarcinoma HT-29 cells, which express only α7-nAChRs. A pronounced antiproliferative effect of both proteins was observed. Incubation of cells with 1 μM SLURP-1 and 1 μM SLURP-2 during 48 h led to a reduction in the cell number down to ~ 54 and 63% relative to the control, respectively. Fluorescent microscopy did not reveal either apoptotic or necrotic cell death. An analysis of the dose-response curve revealed the concentration-dependent mode of the SLURP-1 and SLURP-2 action with EC50 ~ 0.1 and 0.2 nM, respectively. These findings suggest that the α7-nAChR is the main receptor responsible for the antiproliferative effect of SLURP proteins in epithelial cells.

  14. Marrubium vulgare extract inhibits human-LDL oxidation and enhances HDL-mediated cholesterol efflux in THP-1 macrophage.

    Science.gov (United States)

    Berrougui, Hicham; Isabelle, Maxim; Cherki, Mounia; Khalil, Abdelouahed

    2006-12-14

    The objective of the present study was to elucidate the beneficial properties of aqueous extracts of Marrubium vulgare (AEM) towards cardiovascular disease by protecting human-LDL against lipid peroxidation and promoting HDL-mediated cholesterol efflux. Human-LDL were oxidised by incubation with CuSO(4) in the presence of increased concentrations of AEM (0-100 microg/ml). LDL lipid peroxidation was evaluated by conjugated diene formation, vitamin E disappearance as well as LDL-electrophoretic mobility. HDL-mediated cholesterol efflux assay was carried out in human THP-1 macrophages. Incubation of LDL with AEM significantly prolonged the lag phase (P=0.014), lowered the progression rate of lipid peroxidation (P=0.004), reduced the disappearance of vitamin E and the electrophoretic mobility in a dose-dependent manner. Also, incubation of HDL with AEM significantly increased HDL-mediated cholesterol efflux from THP-1 macrophages implicating an independent ATP binding cassette A1 (ABCA1) pathways. Our findings suggest that M. vulgare provides a source of natural antioxidants, which inhibit LDL oxidation and enhance reverse cholesterol transport and thus can prevent cardiovascular diseases development. These antioxidant properties increase the anti-atherogenic potential of HDL.

  15. Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.

  16. Role of α7- and α4β2-nAChRs in the neuroprotective effect of nicotine in stress-induced impairment of hippocampus-dependent memory.

    Science.gov (United States)

    Alzoubi, Karem H; Srivareerat, Marisa; Tran, Trinh T; Alkadhi, Karim A

    2013-06-01

    We have previously shown that nicotine prevents stress-induced memory impairment. In this study, we have investigated the role of α7- and α4β2-nicotinic acetylcholine receptors (nAChRs) in the protective effect of nicotine during chronic stress conditions. Chronic psychosocial stress was induced using a form of rat intruder model. During stress, specific antagonist for either α7-nAChRs [methyllycaconitine (MLA)] or α4β2-nAChRs [dihydro-β-erythroidine (DHβE)] was infused into the hippocampus using a 4-wk osmotic pump at a rate of 82 μg/side.d and 41 μg/side.d, respectively. Three weeks after the start of infusion, all rats were subjected to a series of cognitive tests in the radial arm water maze (RAWM) for six consecutive days or until the animal reached days to criterion (DTC) in the fourth acquisition trial and in all memory tests. DTC is defined as the number of days the animal takes to make no more than one error in three consecutive days. In the short-term memory test, MLA-infused stressed/nicotine-treated rats made similar errors to those of stress and significantly more errors compared to those of stress/nicotine, nicotine or control groups. This finding was supported by the DTC values for the short memory tests. Thus, MLA treatment blocked the neuroprotective effect of nicotine during chronic stress. In contrast, DHβE infusion did not affect the RAWM performance of stress/nicotine animals. These results strongly suggest the involvement of α7-nAChRs, but not α4β2-nAChRs, in the neuroprotective effect of chronic nicotine treatment during chronic stress conditions.

  17. Docking studies of benzylidene anabaseine interactions with α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding proteins (AChBPs): application to the design of related α7 selective ligands.

    Science.gov (United States)

    Kombo, David C; Mazurov, Anatoly; Tallapragada, Kartik; Hammond, Philip S; Chewning, Joseph; Hauser, Terry A; Vasquez-Valdivieso, Montserrat; Yohannes, Daniel; Talley, Todd T; Taylor, Palmer; Caldwell, William S

    2011-11-01

    AChBPs isolated from Lymnaea stagnalis (Ls), Aplysia californica (Ac) and Bulinus truncatus (Bt) have been extensively used as structural prototypes to understand the molecular mechanisms that underlie ligand-interactions with nAChRs [1]. Here, we describe docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR. Results reveal that docking of these compounds using Glide software accurately reproduces experimentally-observed binding modes of DMXBA and of its active metabolite, in the binding pocket of Ac. In addition to the well-known nicotinic pharmacophore (positive charge, hydrogen-bond acceptor, and hydrophobic aromatic groups), a hydrogen-bond donor feature contributes to binding of these compounds to Ac, Bt, and the α7 nAChR. This is consistent with benzylidene anabaseine analogs with OH and NH(2) functional groups showing the highest binding affinity of these congeners, and the position of the ligand shown in previous X-ray crystallographic studies of ligand-Ac complexes. In the predicted ligand-Ls complex, by contrast, the ligand OH group acts as hydrogen-bond acceptor. We have applied our structural findings to optimizing the design of novel spirodiazepine and spiroimidazoline quinuclidine series. Binding and functional studies revealed that these hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype and demonstrate partial agonism. The gain in affinity is also due to conformational restriction, tighter hydrophobic enclosures, and stronger cation-π interactions. The use of AChBPs structure as a surrogate to predict binding affinity to α7 nAChR has also been investigated. On the whole, we found that molecular docking into Ls binding site generally scores better than when a α7 homology model, Bt or Ac crystal structure is used. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  18. Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.

    Science.gov (United States)

    Levenstein, Mark E; Berggren, W Travis; Lee, Ji Eun; Conard, Kevin R; Llanas, Rachel A; Wagner, Ryan J; Smith, Lloyd M; Thomson, James A

    2008-12-01

    Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

  19. Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Chun Zhang; Shibo Tang; Yan Luo; Xiaoling Liang; Jing Ma; Shaofen Lin

    2003-01-01

    Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

  20. Down-regulation of the A3 adenosine receptor in human mast cells upregulates mediators of angiogenesis and remodeling.

    Science.gov (United States)

    Rudich, Noam; Dekel, Ornit; Sagi-Eisenberg, Ronit

    2015-05-01

    Adenosine activated mast cells have been long implicated in allergic asthma and studies in rodent mast cells have assigned the A3 adenosine receptor (A3R) a primary role in mediating adenosine responses. Here we analyzed the functional impact of A3R activation on genes that are implicated in tissue remodeling in severe asthma in the human mast cell line HMC-1 that shares similarities with lung derived human mast cells. Quantitative real time PCR demonstrated upregulation of IL6, IL8, VEGF, amphiregulin and osteopontin. Moreover, further upregulation of these genes was noted upon the addition of dexamethasone. Unexpectedly, activated A3R down regulated its own expression and knockdown of the receptor replicated the pattern of agonist induced gene upregulation. This study therefore identifies the human mast cell A3R as regulator of tissue remodeling gene expression in human mast cells and demonstrates a heretofore-unrecognized mode of feedback regulation that is exerted by this receptor.

  1. Carrier-mediated transport of oligopeptides in the human fibrosarcoma cell line HT1080

    National Research Council Canada - National Science Library

    Nakanishi, T; Tamai, I; Sai, Y; Sasaki, T; Tsuji, A

    1997-01-01

    To explore the feasibility of targeting human tumor cells via their transport systems, dipeptide uptake was studied in the human fibrosarcoma cell line HT1080 and the human fibroblast cell line IMR-90...

  2. A standardized method for quantitating the complement-mediated immune complex solubilizing capacity of human serum

    DEFF Research Database (Denmark)

    Baatrup, G; Peterson, I; Svehag, S E;

    1983-01-01

    A standardized radioassay for measuring the complement-mediated immune complex solubilizing capacity (CMSC) and the initial kinetics of the solubilization (IKS) reaction is described. The total complement (C)-mediated solubilizing capacity was determined after incubation of diluted serum and 125I......-BSA-anti-BSA. Percentage C-mediated solubilization (CMS) was measured after centrifugation by determining the distribution of radioactivity. The dependency of CMSC upon factors such as serum dilution and buffer system used, amount of IC added to serum, serum storage conditions and centrifugation conditions...

  3. Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process.

    Science.gov (United States)

    Ghosal, Abhisek; Sekar, Thillai V; Said, Hamid M

    2014-08-01

    Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na(+)-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na(+)-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS.

  4. Selective inhibition of human heteromeric alpha9alpha10 nicotinic acetylcholine receptors at a low agonist concentration by low concentrations of ototoxic organic solvents.

    Science.gov (United States)

    van Kleef, Regina G D M; Vijverberg, Henk P M; Westerink, Remco H S

    2008-09-01

    Ethylbenzene and para-xylene (p-xylene), but not the chemically closely related organic solvents ortho-xylene (o-xylene) and meta-xylene (m-xylene), are known to cause ototoxicity and irreversible hearing loss, though the underlying mechanisms are still unknown. In this study, effects of ethylbenzene and of p-, o-, and m-xylene on human heteromeric alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated using the two-electrode voltage clamp technique. ACh dose-dependently evoked an alpha9alpha10 nAChR-mediated ion current with an EC(50) of 137 microM. When ACh is applied at a low concentration (10 microM), the nAChR-mediated ion current is inhibited by a low concentration (10 microM) of ethylbenzene and p-xylene, but not by the same concentration of the non-ototoxic solvents. At a high solvent concentration (300 microM), all solvents cause inhibition of the ion currents evoked by 10 microM ACh. Ion currents evoked by a near maximum-effective concentration ACh (1mM) are inhibited by the selected organic solvents only at 300 microM. These results demonstrate that low concentrations of the known ototoxic solvents ethylbenzene and p-xylene inhibit alpha9alpha10 nAChR-mediated ion currents, whereas the structurally related, non-ototoxic solvents m-xylene and o-xylene do not, indicating that the alpha9alpha10 nAChR is a potential target for solvent-induced ototoxicity.

  5. Identification and signature profiles for pro-resolving and inflammatory lipid mediators in human tissue

    National Research Council Canada - National Science Library

    Colas, Romain A; Shinohara, Masakazu; Dalli, Jesmond; Chiang, Nan; Serhan, Charles N

    2014-01-01

    ...(s) to classic eicosanoids in mammalian tissues. Lipid mediator (LM)-SPM levels and signature profiles of their biosynthetic pathways were investigated using liquid chromatography-tandem mass spectrometry (LC-MS-MS...

  6. Evidence for shear stress-mediated dilation of the internal carotid artery in humans

    DEFF Research Database (Denmark)

    Carter, Howard Henry; Atkinson, Ceri L; Heinonen, Ilkka H A

    2016-01-01

    Increases in arterial carbon dioxide tension (hypercapnia) elicit potent vasodilation of cerebral arterioles. Recent studies have also reported vasodilation of the internal carotid artery during hypercapnia, but the mechanism(s) mediating this extracranial vasoreactivity are unknown. Hypercapnia ...

  7. Human xylosyltransferases--mediators of arthrofibrosis? New pathomechanistic insights into arthrofibrotic remodeling after knee replacement therapy

    National Research Council Canada - National Science Library

    Faust, Isabel; Traut, Philipp; Nolting, Frank; Petschallies, Jan; Neumann, Elena; Kunisch, Elke; Kuhn, Joachim; Knabbe, Cornelius; Hendig, Doris

    2015-01-01

    .... We evaluated the association of xylosyltransferases (XT), fibrotic mediators catalyzing glycosaminoglycan biosynthesis, leading to arthrofibrosis as well as the feasibility of using serum XT activity as a diagnostic marker...

  8. Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin

    National Research Council Canada - National Science Library

    Abbot, Emily L; Smith, Wendy D; Siou, Gerard P. S; Chiriboga, Carlos; Smith, Rebecca J; Wilson, Janet A; Hirst, Barry H; Kehoe, Michael A

    2007-01-01

    ... such as Corynebacterium diphtheriae, Streptococcus agalacticae, S. pneumoniae and S. pyogenes. Using various ex vivo tissue and cellular models, here we show that pili mediate adhesion of serotype M1...

  9. Neuropilin-2 mediated β-catenin signaling and survival in human gastro-intestinal cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Shaija Samuel

    Full Text Available NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphorin and vascular endothelial growth factor families. NRP-2 has been detected on the surface of several types of human cancer cells, but its expression and function in gastrointestinal (GI cancer cells remains to be determined. We sought to determine the function of NRP-2 in mediating downstream signals regulating the growth and survival of human gastrointestinal cancer cells. In human gastric cancer specimens, NRP-2 expression was detected in tumor tissues but not in adjacent normal mucosa. In CNDT 2.5 cells, shRNA mediated knockdown NRP-2 expression led to decreased migration and invasion in vitro (p<0.01. Focused gene-array analysis demonstrated that loss of NRP-2 reduced the expression of a critical metastasis mediator gene, S100A4. Steady-state levels and function of β-catenin, a known regulator of S100A4, were also decreased in the shNRP-2 clones. Furthermore, knockdown of NRP-2 sensitized CNDT 2.5 cells in vitro to 5FU toxicity. This effect was associated with activation of caspases 3 and 7, cleavage of PARP, and downregulation of Bcl-2. In vivo growth of CNDT 2.5 cells in the livers of nude mice was significantly decreased in the shNRP-2 group (p<0.05. Intraperitoneal administration of NRP-2 siRNA-DOPC decreased the tumor burden in mice (p = 0.01. Collectively, our results demonstrate that tumor cell-derived NRP-2 mediates critical survival signaling in gastrointestinal cancer cells.

  10. The human septin7 and the yeast CDC10 septin prevent Bax and copper mediated cell death in yeast.

    Science.gov (United States)

    Horowitz, Avital; Lapointe, Jason F; Eid, Rawan; Sheibani, Sara; Gharib, Nada; Jones, Natalie K; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2013-12-01

    The mechanisms of programmed cell death activate genetically encoded intracellular programs in a controlled manner, the most common form being apoptosis. Apoptosis is carried out through a cascade of caspase mediated proteolytic cleavages initiated by the oligomerization of Bax, a cardinal regulator of mitochondrial-mediated apoptosis. Heterologous expression of Bax in yeast causes cell death that shares a number of similarities to processes that occur in mammalian apoptosis. A screen of a cardiac cDNA library for suppressors of Bax-mediated apoptosis identified human septin7, a protein that belongs to the septin superfamily of conserved GTP-binding proteins that share a conserved cdc/septin domain. Analysis of the amino acid sequence deduced from the septin7 clone as well as the corresponding human septin7 gene revealed that a novel alternatively spliced transcript called septin7 variant4 (v4) was uncovered. Yeast cells overexpressing the human septin7 v4 cDNA were also capable of resisting copper-mediated cell death suggesting that it is not only a Bax suppressor but also an anti-apoptotic sequence. Analysis of septin7 function in a MCA1Δ yeast strain suggests that septin7 inhibits apoptosis in a caspase independent pathway. Overexpression of the yeast septin7 ortholog CDC10 also conferred resistance to the negative effects of copper as well as protecting cells from the overexpression of Bax. In contrast, septin7 was unable to prevent the increase in cell size associated with mutants lacking the endogenous yeast CDC10 gene. Taken together, our analysis suggests that anti-apoptosis is a novel yet evolutionarily conserved property of the septin7 sub-family of septins.

  11. Diffuse reflectance spectra measured in vivo in human tissues during Photofrin-mediated pleural photodynamic therapy

    Science.gov (United States)

    Finlay, Jarod C.; Zhu, Timothy C.; Dimofte, Andreea; Friedberg, Joseph S.; Hahn, Stephen M.

    2006-02-01

    Optimal delivery of light in photodynamic therapy (PDT) requires not only optimal placement and power of light sources, but knowledge of the dynamics of light propagation in the tissue being treated and in the surrounding normal tissue, and of their respective accumulations of sensitizer. In an effort to quantify both tissue optical properties and sensitizer distribution, we have measured fluorescence emission and diffuse reflectance spectra at the surface of a variety of tissue types in the thoracic cavities of human patients. The patients studied here were enrolled in Phase II clinical trials of Photofrin-mediated PDT for the treatment of non-small cell lung cancer and cancers with pleural effusion. Patients were given Photofrin at dose of 2 mg per kg body weight 24 hours prior to treatment. Each patient received surgical resection of the affected lung and pleura. Patients received intracavity PDT at 630nm to a dose of 30 J/cm2, as determined by isotropic detectors sutured to the cavity walls. We measured the diffuse reflectance spectra before and after PDT in various positions within the cavity, including tumor, diaphragm, pericardium, skin, and chest wall muscle in 5 patients. The measurements we acquired using a specially designed fiber optic-based probe consisting of one fluorescence excitation fiber, one white light delivery fiber, and 9 detection fibers spaced at distances from 0.36 to 7.8 mm from the source, all of which are imaged via a spectrograph onto a CCD, allowing measurement of radially-resolved diffuse reflectance and fluorescence spectra. The light sources for these two measurements (a 403-nm diode laser and a halogen lamp, respectively) were blocked by computer-controlled shutters, allowing sequential fluorescence, reflectance, and background acquisition. The diffuse reflectance was analyzed to determine the absorption and scattering spectra of the tissue and from these, the concentration and oxygenation of hemoglobin and the local drug uptake

  12. Pharmacological characterization of muscarinic receptor subtypes mediating vasoconstriction of human umbilical vein

    Science.gov (United States)

    Pujol Lereis, Virginia Andrea; Hita, Francisco Javier; Gobbi, Mauro Darío; Verdi, Marcela Gomez; Rodriguez, María Cecilia; Rothlin, Rodolfo Pedro

    2006-01-01

    The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV). HUV rings were mounted in organ baths and concentration–response curves were constructed for acetylcholine (ACh) (pEC50: 6.16±0.04; maximum response 80.00±1.98% of the responses induced by serotonin 10 μM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso-OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC50 6.33±0.03; double inhibition: pEC50 6.57±0.05). Atropine, nonselective muscarinic receptors antagonist, inhibited ACh-induced contraction (pKB 9.67). The muscarinic receptors antagonists pirenzepine (M1), methoctramine (M2) and pFHHSiD (M3) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M4 selective muscarinic receptors antagonist) was ineffective against ACh-induced contraction. In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh-induced responses. The M1 muscarinic receptors agonist McN-A-343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA2 for pirenzepine against McN-A-343 induced responses was 8.54. In conclusion, the data obtained in this study demonstrate the role of M1 muscarinic receptor subtypes and suggest the involvement of M3 muscarinic receptor subtypes in ACh-induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this

  13. High glucose mediates endothelial-to-chondrocyte transition in human aortic endothelial cells

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    Tang Rining

    2012-09-01

    Full Text Available Abstract Background Vascular calcification is one of the common complications in diabetes mellitus. Many studies have shown that high glucose (HG caused cardiovascular calcification, but its underlying mechanism is not fully understood. Recently, medial calcification has been most commonly described in the vessels of patients with diabetes. Chondrocytes were involved in the medial calcification. Recent studies have shown that the conversion into mesenchymal stem cells (MSCs via the endothelial-to-mesenchymal transition (EndMT could be triggered in chondrocytes. Our previous research has indicated that HG induced EndMT in human aortic endothelial cells (HAECs. Therefore, we addressed the question of whether HG-induced EndMT could be transitioned into MSCs and differentiated into chondrocytes. Methods HAECs were divided into three groups: a normal glucose (NG group, HG group (30 mmol/L, and mannitol (5.5 mmol/L NG + 24.5 mmol/L group. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of endothelial markers, such as CD31, and fibroblast markers, such as fibroblast-specific protein 1 (FSP-1. The expression of FSP-1 was detected by real time-PCR and western blots. Endothelial-derived MSCs were grown in MSC medium for one week. The expression of the MSCs markers STRO-1, CD44, CD10 and the chondrocyte marker SOX9 was detected by immunofluorescence staining and western blots. Chondrocyte expression was detected by alcian blue staining. Calcium deposits were analyzed by alizarin red staining. Results The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype. Double staining of the HAECs indicated a co-localization of CD31 and FSP-1. The expression of FSP-1 was significantly increased in the HG group, and the cells undergoing EndMT also expressed STRO-1, CD44 and SOX9 compared with the controls (P  Conclusions Our

  14. Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa

    Science.gov (United States)

    Menegazzo, Massimo; Magagna, Sabina; Di Nisio, Andrea; Šabović, Iva; Rocca, Maria Santa; Scattolini, Valentina; Filippi, Andrea; Foresta, Carlo

    2016-01-01

    The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31–37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis. PMID:27992447

  15. FHL2 mediates tooth development and human dental pulp cell differentiation into odontoblasts, partially by interacting with Runx2.

    Science.gov (United States)

    Du, Jianxin; Wang, Qiang; Yang, Pishan; Wang, Xiaoying

    2016-04-01

    The differentiation of mesenchymal cells in tooth germ and dental pulp cells into odontoblasts is crucial for dentin formation, and the transcription factor runt-related transcription factor (Runx2) is necessary for odontoblast differentiation. Our previous study demonstrated that four and a half LIM domains 2 (FHL2) may play an important role in tooth development and human dental pulp cell differentiation. This study aimed to determine whether FHL2 mediated the mesenchymal cells in tooth development and human dental pulp cell differentiation into odontoblasts by interacting with Runx2. The expression patterns of FHL2 and Runx2 were examined at the early stages of mouse molar development using double immunofluorescence staining. Western blot analysis and co-immunoprecipitation (Co-IP) were conducted for the preliminary study of the relationship between FHL2 and Runx2 in human dental pulp cell differentiation into odontoblasts. Results of double immunofluorescence staining showed that FHL2 and Runx2 exhibited similar expression patterns at the early stages of tooth development. Western blot analysis indicated that the expression patterns of FHL2 and Runx2 were synchronized on day 7 of induction, whereas those on day 14 differed. Co-IP analysis revealed positive bands of protein complexes, revealing the interaction of FHL2 and Runx2 on days 0, 7 and 14 of induction. Our data suggested that FHL2 might interact with Runx2 to mediate mesenchymal cell differentiation at the early stages of tooth development and human dental pulp cell differentiation.

  16. 6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Igor E. Kasheverov

    2015-03-01

    Full Text Available 6-Bromohypaphorine (6-BHP has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR. Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM, but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM. To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.

  17. The human and mouse receptors of hyaluronan-mediated motility, RHAMM, genes (HMMR) map to human chromosome 5q33.2-qter and mouse chromosome 11

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Roller, M.L.; Camper, S.A. [Univ. of Michigan Medical School, Ann Arbor, MI (United States)] [and others

    1995-11-01

    The gene for the receptor for hyaluronan-mediated motility, RHAAM (designated hyaluronan-mediated motility receptor, HMMR (human) and Hmmr (mouse), for mapping purposes), was localized to human chromosome 5q33.2-qter by somatic cell and radiation hybrid analyses. Investigation of two interspecific back-crosses localized the mouse RHAMM (Hmmr) locus 18 cM from the centromere of mouse chromosome 11 within a region of synteny homology with human chromosome 5q23-q35 genes. The map position of the human RHAMM gene places it in a region comparatively rich in disease-associated genes, including those for low-frequency hearing loss, dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q-syndrome. The RHAMM gene location and its ability to transform cells when overexpressed implicate RHAMM as a possible candidate gene in the pathogenesis of the recently described t(5;14)(q33-q34;q11) acute lymphoblastic leukemias. 18 refs., 1 fig.

  18. SIGIRR inhibits toll-like receptor 4, 5, 9-mediated immune responses in human airway epithelial cells.

    Science.gov (United States)

    Zhang, Chun; Wu, Xueling; Zhao, Yunfeng; Deng, Zhaoxia; Qian, Guisheng

    2011-01-01

    Human airway epithelial cells (HAEC) may contribute to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) through toll-like receptors (TLRs)-mediated molecular mechanisms. TLRs exist on the surface of HAEC where binding to their cognate ligands initiates airway inflammation. Single immunoglobulin interleukin-1 receptor-related protein (SIGIRR) is a member of the toll-interleukin-1 receptor (TIR) family that can negatively modulate the immune response. We carried out studies to characterize SIGIRR modulation of TLR-mediated immune response in HAEC and to define its mechanisms of action. Following treatment with various concentrations of LPS, flagellin and CpG DNA, the levels of cognate TLRs 4, 5, and 9 were measured in the supernatants of HAEC over-expressing the SIGIRR molecule. Moreover, the interaction of the TLR adaptor myeloid differentiation factor 88 (MyD88) with SIGIRR in response to LPS-, flagellin- and CpG DNA-stimulation was examined by co-immunoprecipitation. The findings from this study revealed that overexpression of SIGIRR in HAEC stimulated by LPS, flagellin or CpG DNA resulted in attenuated production of the inflammatory mediators IL-6 and TNF-α. This attenuation was not the result of decreased expression of TLR4, 5 or 9, but rather a sequestration of MyD88 to the TLRs. In conclusion, SIGIRR can inhibit TLR4, 5, and 9-mediated immune responses in HAEC and may be a valuable therapeutic target for the prevention of ALI/ARDS.

  19. Nicotine Induces the Production of IL-1ß and IL-8 via the a7 nAChR/NF-κB Pathway in Human Periodontal Ligament Cells: an in Vitro Study

    Directory of Open Access Journals (Sweden)

    Lizheng Wu

    2014-07-01

    Full Text Available Background/Aims: Tobacco smoking is a major risk factor for the occurrence and progression of periodontitis. We previously demonstrated that nicotine could induce the expression of a7 nicotinic acetylcholine receptors (a7 nAChR in human and rat periodontal tissues. To further examine the signal pathways mediated by a7 nAChR in periodontal ligament (PDL cells, we investigated whether nicotine affects interleukin-1ß (IL-1ß and interleukin-8 (IL-8 via the a7 nAChR/NF-κB pathway in human PDL cells. Methods: Human PDL cells were pre-incubated with alpha-bungarotoxin (a-BTX or pyrrolidine dithiocarbamate (PDTC, then cultured with nicotine. Then, we used western blotting, a dual-luciferase reporter, real-time quantitative PCR and an enzyme-linked immunoassay to assess expression of the NF-κB p65 subunit, NF-κB activity and production of IL-1ß and IL-8 in human PDL cells. Results: Compared with the control group, nicotine could significantly induce production of IL-1ß and IL-8 in human PDL cells and cause the similar effects on the expression of the NF-κB p65 subunit and NF-κB activity. Conclusion: This study demonstrates that nicotine could induce production of IL-1ß and IL-8 via the a7 nAChR/NF-κB pathway in human PDL cells, providing data for a better understanding of the relationships among smoking, nicotine, and periodontitis.

  20. Nicotine induces the production of IL-1β and IL-8 via the α7 nAChR/NF-κB pathway in human periodontal ligament cells: an in vitro study.

    Science.gov (United States)

    Wu, Lizheng; Zhou, Yongchuan; Zhou, Zhifei; Liu, Yingfeng; Bai, Yudi; Xing, Xianghui; Wang, Xiaojing

    2014-01-01

    Tobacco smoking is a major risk factor for the occurrence and progression of periodontitis. We previously demonstrated that nicotine could induce the expression of α7 nicotinic acetylcholine receptors (α7 nAChR) in human and rat periodontal tissues. To further examine the signal pathways mediated by α7 nAChR in periodontal ligament (PDL) cells, we investigated whether nicotine affects interleukin-1β (IL-1β) and interleukin-8 (IL-8) via the α7 nAChR/NF-κB pathway in human PDL cells. Human PDL cells were pre-incubated with alpha-bungarotoxin (α-BTX) or pyrrolidine dithiocarbamate (PDTC), then cultured with nicotine. Then, we used western blotting, a dual-luciferase reporter, real-time quantitative PCR and an enzyme-linked immunoassay to assess expression of the NF-κB p65 subunit, NF-κB activity and production of IL-1β and IL-8 in human PDL cells. Compared with the control group, nicotine could significantly induce production of IL-1β and IL-8 in human PDL cells and cause the similar effects on the expression of the NF-κB p65 subunit and NF-κB activity. This study demonstrates that nicotine could induce production of IL-1β and IL-8 via the α7 nAChR/NF-κB pathway in human PDL cells, providing data for a better understanding of the relationships among smoking, nicotine, and periodontitis. © 2014 S. Karger AG, Basel.

  1. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.

    Science.gov (United States)

    Wang, Yebo; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

    2016-11-28

    Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.

  2. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hanwen [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States); Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, University of South Carolina, Columbia, SC 29208 (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States)

    2015-01-01

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.

  3. Suppression of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells.

    Science.gov (United States)

    Sehrawat, Anuradha; Kim, Su-Hyeong; Vogt, Andreas; Singh, Shivendra V

    2013-04-01

    We showed previously that breast cancer chemoprevention with benzyl isothiocyanate (BITC) in MMTV-neu mice was associated with induction of E-cadherin protein in vivo. Loss of E-cadherin expression and induction of mesenchymal markers (e.g. vimentin) are biochemical hallmarks of epithelial-mesenchymal transition (EMT), a developmental process implicated in progression of cancer to aggressive state. This study offers novel insights into the mechanism by which BITC inhibits EMT. Exposure of MDA-MB-231, SUM159 and MDA-MB-468 human breast cancer cells to BITC (2.5 and 5 µM) resulted in transcriptional repression of urokinase-type plasminogen activator (uPA) as well as its receptor (uPAR). However, ectopic expression of uPAR in MDA-MB-468 cells failed to confer protection against induction of E-cadherin and inhibition of cell invasion/migration resulting from BITC treatment. The BITC-mediated induction of E-cadherin and inhibition of cell migration was sustained in MDA-MB-231 and SUM159 cells transiently transfected with an uPAR-targeted small interfering RNA. Overexpression of Forkhead Box Q1 (FOXQ1), whose protein and messenger RNA levels were decreased by BITC treatment in cells and MDA-MB-231 xenografts, conferred marked protection against BITC-mediated inhibition of EMT and cell migration. In conclusion, this study implicates FOXQ1 suppression in BITC-mediated inhibition of EMT in human breast cancer cells.

  4. HSV-1 amplicon-mediated post-transcriptional inhibition of Rad51 sensitizes human glioma cells to ionizing radiation.

    Science.gov (United States)

    Saydam, O; Saydam, N; Glauser, D L; Pruschy, M; Dinh-Van, V; Hilbe, M; Jacobs, A H; Ackermann, M; Fraefel, C

    2007-08-01

    Standard treatment for glioblastoma multiforme and other brain tumors consists of surgical resection followed by combined radio-/chemotherapy. However, radiation resistance of tumor cells limits the success of this treatment, and the tumors invariably recur. Therefore, the selective inhibition of molecular mediators of radiation resistance may provide therapeutic benefit to the patient. One of these targets is the Rad51 protein, which is a key component of the homologous recombinational repair of DNA double-strand breaks. Here, we investigated whether post-transcriptional silencing of Rad51 by herpes simplex virus-type 1 (HSV-1) amplicon vector-mediated short interfering RNA expression can enhance the antitumor effect of radiation therapy. We demonstrate that these vectors specifically and efficiently inhibited the radiation-induced recruitment of Rad51 into nuclear foci in human glioma cells. The combination of vector-mediated silencing of Rad51 expression and treatment with ionizing radiation resulted in a pronounced reduction of the survival of human glioma cells in culture. In athymyc mice, a single intratumoral injection of Rad51-specific HSV-1 amplicon vector followed by a single radiation treatment resulted in a significant decrease in tumor size. In control animals, including mice that received an intratumoral injection of Rad51-specific amplicon vector but no radiation treatment, the tumor sizes increased.

  5. The relationship between human resource development factors, career growth and turnover intention: The mediating role of organizational commitment

    Directory of Open Access Journals (Sweden)

    Muhammad Shahid Nawaz

    2016-02-01

    Full Text Available Retaining the best employees is of high concern for most organizations and this issue has become a significant focus of attention for many researchers. For this reason, this paper discusses different factors which influence the employee turnover intention-behavior in the organization, specifically to examine the effect of salary, performance appraisal, training & development and career growth on turnover intention. In addition, based on the social exchange theory this paper explains the mediating role of organizational commitment in the relationship between human resource development factors, career growth and turnover intention. A cross sectional, survey data study is undertaken to investigate the relationships in a sample of 270 full time faculty members employed in different private universities of Pakistan. Partial Least Square two step path modeling is used to test the direct and the indirect hypothesis of the study. The results of PLS (SEM path modeling reveal that human resource development factors specially salary and performance appraisal were negatively associated with turnover intention. In addition, the results also indicate that career growth had significant relationships with turnover intention. Moreover, out of four dimensions of career growth, only two dimensions, namely promotion speed and remuneration growth, have strong influence on turnover intention. Finally, in terms of organizational commitment as mediating variable between the relationships of salary, performance appraisal, career growth and turnover intention, four out of six variables indicate partial mediation including career growth (career goal progress, career growth (promotion speed, career growth (remuneration growth and performance appraisal.

  6. Effect of miR-451-mediated regulation of MIF expression on cell proliferation in human colon carcinoma cell line LoVo

    Institute of Scientific and Technical Information of China (English)

    孔帅

    2013-01-01

    Objective To investigate the effect of miR-451-mediated regulation of macrophage migration inhibitory factor(MIF) expression on cell proliferation in human colon carcinoma cell line LoVo.Methods A lentiviral vector

  7. Post-operative infection and sepsis in humans is associated with deficient gene expression of γc cytokines and their apoptosis mediators.

    LENUS (Irish Health Repository)

    White, Mary

    2011-06-01

    Lymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators.

  8. Human papillomavirus-mediated carcinogenesis and HPV-associated oral and oropharyngeal squamous cell carcinoma. Part 1: Human papillomavirus-mediated carcinogenesis

    Directory of Open Access Journals (Sweden)

    Khammissa Razia AG

    2010-07-01

    Full Text Available Abstract High-risk human papillomavirus (HPV E6 and E7 oncoproteins are essential factors for HPV-induced carcinogenesis, and for the maintenance of the consequent neoplastic growth. Cellular transformation is achieved by complex interaction of these oncogenes with several cellular factors of cell cycle regulation including p53, Rb, cyclin-CDK complexes, p21 and p27. Both persistent infection with high-risk HPV genotypes and immune dysregulation are associated with increased risk of HPV-induced squamous cell carcinoma.

  9. Cocaine exposure impairs multilineage hematopoiesis of human hematopoietic progenitor cells mediated by the sigma-1 receptor [corrected].

    Science.gov (United States)

    Nixon, Christopher C; Schwartz, Brandon H; Dixit, Dhaval; Zack, Jerome A; Vatakis, Dimitrios N

    2015-03-02

    Prenatal exposure to cocaine is a significant source of fetal and neonatal developmental defects. While cocaine associated neurological and cardiac pathologies are well-documented, it is apparent that cocaine use has far more diverse physiological effects. It is known that in some cell types, the sigma-1 receptor mediates many of cocaine's cellular effects. Here we present a novel and concise investigation into the mechanism that underlies cocaine associated hematopoietic pathology. Indeed, this is the first examination of the effects of cocaine on hematopoiesis. We show that cocaine impairs multilineage hematopoiesis from human progenitors from multiple donors and tissue types. We go on to present the first demonstration of the expression of the sigma-1 receptor in human CD34 + human hematopoietic stem/progenitor cells. Furthermore, we demonstrate that these cocaine-induced hematopoietic defects can be reversed through sigma-1 receptor blockade.

  10. Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1.

    Science.gov (United States)

    Campbell, Grant R; Spector, Stephen A

    2012-01-01

    Toll-like receptors (TLR) are important in recognizing microbial pathogens and triggering host innate immune responses, including autophagy, and in the mediation of immune activation during human immunodeficiency virus type-1 (HIV) infection. We report here that TLR8 activation in human macrophages induces the expression of the human cathelicidin microbial peptide (CAMP), the vitamin D receptor (VDR) and cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1), which 1α-hydroxylates the inactive form of vitamin D, 25-hydroxycholecalciferol, into its biologically active metabolite. Moreover, we demonstrate using RNA interference, chemical inhibitors and vitamin D deficient media that TLR8 agonists inhibit HIV through a vitamin D and CAMP dependent autophagic mechanism. These data support an important role for vitamin D in the control of HIV infection, and provide a biological explanation for the benefits of vitamin D. These findings also provide new insights into potential novel targets to prevent and treat HIV infection.

  11. The anti-inflammatory effects of PGE2 on human lung macrophages are mediated by the EP4 receptor.

    Science.gov (United States)

    Gill, Sharonjit K; Yao, Yiwen; Kay, Linda J; Bewley, Martin A; Marriott, Helen M; Peachell, Peter T

    2016-11-01

    PGE2 inhibits cytokine generation from human lung macrophages. However, the EP receptor that mediates this beneficial anti-inflammatory effect of PGE2 has not been defined. The aim of this study was to identify the EP receptor by which PGE2 inhibits cytokine generation from human lung macrophages. This was determined by using recently developed EP receptor ligands. The effects of PGE2 and EP-selective agonists on LPS-induced generation of TNF-α and IL-6 from macrophages were evaluated. The effects of EP2 -selective (PF-04852946, PF-04418948) and EP4 -selective (L-161,982, CJ-042794) receptor antagonists on PGE2 responses were studied. The expression of EP receptor subtypes by human lung macrophages was determined by RT-PCR. PGE2 inhibited LPS-induced and Streptococcus pneumoniae-induced cytokine generation from human lung macrophages. Analysis of mRNA levels indicated that macrophages expressed EP2 and EP4 receptors. L-902,688 (EP4 receptor-selective agonist) was considerably more potent than butaprost (EP2 receptor-selective agonist) as an inhibitor of TNF-α generation from macrophages. EP2 receptor-selective antagonists had marginal effects on the PGE2 inhibition of TNF-α generation, whereas EP4 receptor-selective antagonists caused rightward shifts in the PGE2 concentration-response curves. These studies demonstrate that the EP4 receptor is the principal receptor that mediates the anti-inflammatory effects of PGE2 on human lung macrophages. This suggests that EP4 receptor agonists could be effective anti-inflammatory agents in human lung disease. © 2016 The British Pharmacological Society.

  12. The PPAR alpha-humanized mouse: a model to investigate species differences in liver toxicity mediated by PPAR alpha.

    Science.gov (United States)

    Yang, Qian; Nagano, Tomokazu; Shah, Yatrik; Cheung, Connie; Ito, Shinji; Gonzalez, Frank J

    2008-01-01

    To determine the impact of the species difference between rodents and humans in response to peroxisome proliferators (PPs) mediated by peroxisome proliferator-activated receptor (PPAR)alpha, PPAR alpha-humanized transgenic mice were generated using a P1 phage artificial chromosome (PAC) genomic clone bred onto a ppar alpha-null mouse background, designated hPPAR alpha PAC. In hPPAR alpha PAC mice, the human PPAR alpha gene is expressed in tissues with high fatty acid catabolism and induced upon fasting, similar to mouse PPAR alpha in wild-type (Wt) mice. Upon treatment with the PP fenofibrate, hPPAR alpha PAC mice exhibited responses similar to Wt mice, including peroxisome proliferation, lowering of serum triglycerides, and induction of PPAR alpha target genes encoding enzymes involved in fatty acid metabolism in liver, kidney, and heart, suggesting that human PPAR alpha (hPPAR alpha) functions in the same manner as mouse PPAR alpha in regulating fatty acid metabolism and lowering serum triglycerides. However, in contrast to Wt mice, treatment of hPPAR alpha PAC mice with fenofibrate did not cause significant hepatomegaly and hepatocyte proliferation, thus indicating that the mechanisms by which PPAR alpha affects lipid metabolism are distinct from the hepatocyte proliferation response, the latter of which is only induced by mouse PPAR alpha. In addition, a differential regulation of several genes, including the oncogenic let-7C miRNA by PPs, was observed between Wt and hPPAR alpha PAC mice that may contribute to the inherent difference between mouse and human PPAR alpha in activation of hepatocellular proliferation. The hPPAR alpha PAC mouse model provides an in vivo platform to investigate the species difference mediated by PPAR alpha and an ideal model for human risk assessment PPs exposure.

  13. Clarifying the Effects of Human Resource Diversity Management Practices on Organizational Citizenship Behavior: The Mediating Role of Diversity Receptiveness

    Directory of Open Access Journals (Sweden)

    Ahmad Nizan Mat Noor

    2014-06-01

    Full Text Available The aim of this study is to scrutinize the impact of employees’ perceptions of their organization’s human resource diversity management (HRDM practices on their organizational citizenship behavior (OCB level. The influence of diversity receptiveness as a mediator in the proposed relationship is also examined. Survey data were gathered from operational employees attached to large hotel in Malaysia. 430 usable questionnaires were used in statistical analyses. The results indicated that the hypothesized linkage between HRDM practices and diversity receptiveness as well as between HRDM practices and OCB were partially supported. The mediating role of diversity receptiveness in the relationship was also partially supported. Implications and limitations of the findings are specified. Finally, directions for future research are suggested.

  14. Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

    Science.gov (United States)

    Li, Xiaowei; Tzeng, Stephany Y; Liu, Xiaoyan; Tammia, Markus; Cheng, Yu-Hao; Rolfe, Andrew; Sun, Dong; Zhang, Ning; Green, Jordan J; Wen, Xuejun; Mao, Hai-Quan

    2016-04-01

    Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy.

  15. Rac1 regulates the NLRP3 inflammasome which mediates IL-1beta production in Chlamydophila pneumoniae infected human mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Julia Eitel

    Full Text Available Chlamydophila pneumoniae causes acute respiratory tract infections and has been associated with development of asthma and atherosclerosis. The production of IL-1β, a key mediator of acute and chronic inflammation, is regulated on a transcriptional level and additionally on a posttranslational level by inflammasomes. In the present study we show that C. pneumoniae-infected human mononuclear cells produce IL-1β protein depending on an inflammasome consisting of NLRP3, the adapter protein ASC and caspase-1. We further found that the small GTPase Rac1 is activated in C. pneumoniae-infected cells. Importantly, studies with specific inhibitors as well as siRNA show that Rac1 regulates inflammasome activation in C. pneumoniae-infected cells. In conclusion, C. pneumoniae infection of mononuclear cells stimulates IL-1β production dependent on a NLRP3 inflammasome-mediated processing of proIL-1β which is controlled by Rac1.

  16. Too Human versus the enthusiast press: Video game journalists as mediators of commodity value

    Directory of Open Access Journals (Sweden)

    Rebecca Carlson

    2009-03-01

    Full Text Available This article examines the role of mediators in the production of commodity value, arguing that there is a rise in a kind of immaterial labor, shaped by contemporary conditions of late capitalism, that functions explicitly as a mediating force. In this example, video game journalists are understood as actively engaged in producing and negotiating the value and meaning of video games for both producers and consumers. By specifically examining a moment of value contestation, a podcast debate between a journalist and a game developer, this article traces the mediating practices of the enthusiast gaming press and examines the way their history with and relationship to the video game industry continue to structure their ability to filter knowledge and shape desires.

  17. Too Human versus the enthusiast press: Video game journalists as mediators of commodity value

    Directory of Open Access Journals (Sweden)

    Rebecca Carlson

    2009-03-01

    Full Text Available This article examines the role of mediators in the production of commodity value, arguing that there is a rise in a kind of immaterial labor, shaped by contemporary conditions of late capitalism, that functions explicitly as a mediating force. In this example, video game journalists are understood as actively engaged in producing and negotiating the value and meaning of video games for both producers and consumers. By specifically examining a moment of value contestation, a podcast debate between a journalist and a game developer, this article traces the mediating practices of the enthusiast gaming press and examines the way their history with and relationship to the video game industry continue to structure their ability to filter knowledge and shape desires.

  18. TRIM32 promotes retinoic acid receptor {alpha}-mediated differentiation in human promyelogenous leukemic cell line HL60

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Tomonobu [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Okumura, Fumihiko [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Iguchi, Akihiro; Ariga, Tadashi [Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Hatakeyama, Shigetsugu, E-mail: hatas@med.hokudai.ac.jp [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer TRIM32 enhanced RAR{alpha}-mediated transcriptional activity even in the absence of RA. Black-Right-Pointing-Pointer TRIM32 stabilized RAR{alpha} in the human promyelogenous leukemic cell line HL60. Black-Right-Pointing-Pointer Overexpression of TRIM32 in HL60 cells induced granulocytic differentiation. Black-Right-Pointing-Pointer TRIM32 may function as a coactivator for RAR{alpha}-mediated transcription in APL cells. -- Abstract: Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor {alpha} (RAR{alpha}). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RAR{alpha} and enhances transcriptional activity of RAR{alpha} in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RAR{alpha}, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RAR{alpha}-mediated transcriptional activity even in the absence of RA and stabilizes RAR{alpha} in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RAR{alpha}-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

  19. Human immunodeficiency virus type 1 infection of human uterine epithelial cells: viral shedding and cell contact-mediated infectivity.

    Science.gov (United States)

    Asin, Susana N; Wildt-Perinic, Dunja; Mason, Sarah I; Howell, Alexandra L; Wira, Charles R; Fanger, Michael W

    2003-05-15

    We examined the mechanism of human immunodeficiency virus (HIV) type 1 infection of human uterine epithelial cells to gain a clearer understanding of the events by which HIV-1 infects cells within the female reproductive tract. We demonstrated that these cells can be productively infected by HIV-1 and that infection is associated with viral RNA reverse transcription, DNA transcription, and secretion of infectious virus. Levels of viral DNA and secreted virus decreased gradually after infection. Moreover, virus released by the uterine epithelial cells shortly after infection was able to infect human T cell lines, but virus released later did not. In contrast, human CD4(+) T cell lines were infected after cocultivation with epithelial cells at both early and late stages of infection. These data demonstrated that HIV-1 infects human epithelial cells of upper reproductive tract origin and that productive viral infection of epithelial cells may be an important mechanism of transmission of HIV-1 infection in women.

  20. Observational evidence for human impact on aerosol cloud-mediated processes in the Baltic region

    Directory of Open Access Journals (Sweden)

    Olaf Krüger

    2014-03-01

    Full Text Available Knowledge about aerosol cloud-mediated processes is important for judging climate change in Europe during recent decades. Here, some observational evidence for anthropogenic influences is described and discussed. The emphasis is laid on the effects of the large emissions of sulphur dioxide and particulate matter during the 1980s in Europe and the subsequent strong decrease in the 1990s. In addition, an analysis of the dependence of aerosol cloud-mediated processes on atmospheric circulation patterns (Grosswetterlagen is presented.

  1. Observational evidence for human impact on aerosol cloud-mediated processes in the Baltic region

    Directory of Open Access Journals (Sweden)

    Olaf Krüger

    2014-01-01

    Full Text Available Knowledge about aerosol cloud-mediated processes is important for judging climate change in Europe during recent decades. Here, some observational evidence for anthropogenic influences is described and discussed. The emphasis is laid on the effects of the large emissions of sulphur dioxide and particulate matter during the 1980s in Europe and the subsequent strong decrease in the 1990s. In addition, an analysis of the dependence of aerosol cloud-mediated processes on atmospheric circulation patterns (Großwetterlagen is presented.

  2. Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3.

    Science.gov (United States)

    Gu, Shen; Yuan, Bo; Campbell, Ian M; Beck, Christine R; Carvalho, Claudia M B; Nagamani, Sandesh C S; Erez, Ayelet; Patel, Ankita; Bacino, Carlos A; Shaw, Chad A; Stankiewicz, Paweł; Cheung, Sau Wai; Bi, Weimin; Lupski, James R

    2015-07-15

    Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.

  3. Alteration of Cell Cycle Mediated by Zinc in Human Bronchial Epithelial Cells In Vitro

    Science.gov (United States)

    Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examin...

  4. Alteration of Cell Cycle Mediated by Zinc in Human Bronchial Epithelial Cells In Vitro

    Science.gov (United States)

    Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examin...

  5. Amphiregulin mediates hCG-induced StAR expression and progesterone production in human granulosa cells

    Science.gov (United States)

    Fang, Lanlan; Yu, Yiping; Zhang, Ruizhe; He, Jingyan; Sun, Ying-Pu

    2016-01-01

    Progesterone plays critical roles in maintaining a successful pregnancy at the early embryonic stage. Human chorionic gonadotropin (hCG) rapidly induces amphiregulin (AREG) expression. However, it remains unknown whether AREG mediates hCG-induced progesterone production. Thus, the objective of this study was to investigate the role of AREG in hCG-induced progesterone production and the underlying molecular mechanism in human granulosa cells; primary cells were used as the experimental model. We demonstrated that the inhibition of EGFR and the knockdown of AREG abolished hCG-induced steroidogenic acute regulatory protein (StAR) expression and progesterone production. Importantly, follicular fluid AREG levels were positively correlated with progesterone levels in the follicular fluid and serum. Treatment with AREG increased StAR expression and progesterone production, and these stimulatory effects were abolished by EGFR inhibition. Moreover, activation of ERK1/2, but not PI3K/Akt, signaling was required for the AREG-induced up-regulation of StAR expression and progesterone production. Our results demonstrate that AREG mediates hCG-induced StAR expression and progesterone production in human granulosa cells, providing novel evidence for the role of AREG in the regulation of steroidogenesis. PMID:27113901

  6. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.

    Science.gov (United States)

    Zhang, Yilan; Ge, Xianglian; Yang, Fayu; Zhang, Liping; Zheng, Jiayong; Tan, Xuefang; Jin, Zi-Bing; Qu, Jia; Gu, Feng

    2014-06-23

    CRISPR/Cas9-mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the human EMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quantitatively test the efficiency of CCMDC with non-canonical PAMs in human cells. The initial results indicated that the effectiveness of NGA PAM for CCMDC is much higher than that of other 14 PAMs including NAG. Then we further designed another three pairs of NGG, NGA and NAG PAMs at different locations in the GFP gene and investigated the corresponding DNA cleavage efficiency. We observed that one group of NGA PAMs have a relatively higher DNA cleavage efficiency, while the other groups have lower efficiency, compared with the corresponding NAG PAMs. Our study clearly demonstrates that NAG may not be the universally predominant non-canonical PAM for CCMDC in human cells. These findings raise more concerns over off-target effects in CRISPR/Cas9-mediated genome engineering.

  7. FLIP switches Fas-mediated glucose signaling in human pancreatic β cells from apoptosis to cell replication

    Science.gov (United States)

    Maedler, Kathrin; Fontana, Adriano; Ris, Frédéric; Sergeev, Pavel; Toso, Christian; Oberholzer, José; Lehmann, Roger; Bachmann, Felix; Tasinato, Andrea; Spinas, Giatgen A.; Halban, Philippe A.; Donath, Marc Y.

    2002-01-01

    Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic β cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of β cell turnover. In human islets, elevated glucose concentrations impair β cell proliferation and induce β cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic β cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive β cells; FLIP was no longer detectable in such TUNEL-positive β cells. Up-regulation of FLIP, by incubation with transforming growth factor β or by transfection with an expression vector coding for FLIP, protected β cells from glucose-induced apoptosis, restored β cell proliferation, and improved β cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human β cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation. PMID:12060768

  8. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    Science.gov (United States)

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.

  9. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    Energy Technology Data Exchange (ETDEWEB)

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. (Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill (United States))

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  10. A type-II positive allosteric modulator of α7 nAChRs reduces brain injury and improves neurological function after focal cerebral ischemia in rats.

    Directory of Open Access Journals (Sweden)

    Fen Sun

    Full Text Available In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II of α7 nicotinic acetylcholine receptors (nAChRs, as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min middle cerebral artery occlusion (MCAO. Animals were then subdivided into two groups and injected intravenously (i.v. 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group or vehicle only (untreated group. Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.

  11. Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Javed [Department of Zoology, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Ahamed, Maqusood, E-mail: maqusood@gmail.com [King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451 (Saudi Arabia); Akhtar, Mohd Javed [Fibre Toxicology, CSIR-Indian Institute of Toxicology Research, Lucknow-226001 (India); Alrokayan, Salman A. [King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451 (Saudi Arabia); Siddiqui, Maqsood A.; Musarrat, Javed; Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia)

    2012-03-01

    Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25–200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion of glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level. -- Highlights: ► We explored the mechanisms of toxicity caused by silica NPs in human liver HepG2 cells. ► Silica NPs induced a dose-dependent cytotoxicity in HepG2 cells. ► Silica NPs induced ROS generation and oxidative stress in a dose-dependent manner. ► Silica NPs were also modulated apoptosis markers both at mRNA and protein levels. ► ROS mediated apoptosis induced by silica NPs was preserved by vitamin C.

  12. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    Science.gov (United States)

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.

  13. Epinephrine mediates facultative carbohydrate-induced thermogenesis in human skeletal muscle

    DEFF Research Database (Denmark)

    Astrup, A; Simonsen, L; Bülow, J

    1989-01-01

    The thermic effect of carbohydrate has a component mediated by the sympathoadrenal system but of unknown anatomical localization. We have studied the contribution of skeletal muscle to the thermic effect of a carbohydrate-rich natural meal (115 g of carbohydrate, approximately 80% of energy) by m...

  14. Caffeine's impairment of insulin-mediated glucose disposal cannot be solely attributed to adrenaline in humans

    DEFF Research Database (Denmark)

    Battram, D S; Graham, T E; Dela, F

    2007-01-01

    Caffeine (CAF) impedes insulin-mediated glucose disposal (IMGD) and increases plasma adrenaline concentrations ([ADR]; 0.6 nm). While the antagonism of ADR abolishes the CAF effect, infusion of ADR (0.75 nm) has no effect on IMGD. We have now examined CAF and ADR in concert to determine whether...

  15. Lipid mediators involved in the oxidative stress and antioxidant defence of human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Agnieszka Gęgotek

    2016-10-01

    Conclusions: This study shows significant differences in the redox status, Nrf2 pathway and endocannabinoid system between SCC and AC tissues. Understanding the relation between the various lipid mediators and antioxidants in different lung cancer subtypes may be beginning for further research on the effective anticancer therapy.

  16. Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in alloimmunized baboons

    NARCIS (Netherlands)

    Tillou, Xavier; Poirier, Nicolas; Le Bas-Bernardet, Stephanie; Hervouet, Jeremy; Minault, David; Renaudin, Karine; Vistoli, Fabio; Karam, Georges; Daha, Mohamed; Soulillou, Jean Paul; Blancho, Gilles

    2010-01-01

    Acute antibody-mediated rejection is an unsolved issue in transplantation, especially in the context of pretransplant immunization. The deleterious effect of preformed cytotoxic anti-HLA antibodies through complement activation is well proven, but very little is known concerning complement blockade

  17. Wild-Type, but Not Mutant N296H, Human Tau Restores Aβ-Mediated Inhibition of LTP in Tau−/− mice

    Directory of Open Access Journals (Sweden)

    Mariana Vargas-Caballero

    2017-04-01

    Full Text Available Microtubule associated protein tau (MAPT is involved in the pathogenesis of Alzheimer's disease and many forms of frontotemporal dementia (FTD. We recently reported that Aβ-mediated inhibition of hippocampal long-term potentiation (LTP in mice requires tau. Here, we asked whether expression of human MAPT can restore Aβ-mediated inhibition on a mouse Tau−/− background and whether human tau with an FTD-causing mutation (N296H can interfere with Aβ-mediated inhibition of LTP. We used transgenic mouse lines each expressing the full human MAPT locus using bacterial artificial chromosome technology. These lines expressed all six human tau protein isoforms on a Tau−/− background. We found that the human wild-type MAPT H1 locus was able to restore Aβ42-mediated impairment of LTP. In contrast, Aβ42 did not reduce LTP in slices in two independently generated transgenic lines expressing tau protein with the mutation N296H associated with frontotemporal dementia (FTD. Basal phosphorylation of tau measured as the ratio of AT8/Tau5 immunoreactivity was significantly reduced in N296H mutant hippocampal slices. Our data show that human MAPT is able to restore Aβ42-mediated inhibition of LTP in Tau−/− mice. These results provide further evidence that tau protein is central to Aβ-induced LTP impairment and provide a valuable tool for further analysis of the links between Aβ, human tau and impairment of synaptic function.

  18. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells

    OpenAIRE

    Yilan Zhang; Xianglian Ge; Fayu Yang; Liping Zhang; Jiayong Zheng; Xuefang Tan; Zi-Bing Jin; Jia Qu; Feng Gu

    2014-01-01

    CRISPR/Cas9 -mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the human EMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quanti...

  19. Insight into the Mechanism of Human Herpesvirus 7 U21-mediated Diversion of Class I MHC Molecules to Lysosomes*

    Science.gov (United States)

    Glosson, Nicole L.; Gonyo, Patrick; May, Nathan A.; Schneider, Christine L.; Ristow, Laura C.; Wang, Qiuhong; Hudson, Amy W.

    2010-01-01

    The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules. PMID:20833720

  20. Expression of Glycosylphosphatidylinositol-Anchored CD59 on Target Cells Enhances Human NK Cell-Mediated Cytotoxicity1

    OpenAIRE

    2006-01-01

    NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs...

  1. Expression of a humanized viral 2A-mediated lux operon efficiently generates autonomous bioluminescence in human cells.

    Directory of Open Access Journals (Sweden)

    Tingting Xu

    Full Text Available Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines.The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations.Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity

  2. CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

    Science.gov (United States)

    Mende, Nicole; Kuchen, Erika E; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico; Waskow, Claudia

    2015-07-27

    Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. © 2015 Mende et al.

  3. EFFECT OF ADENOVIRUS-MEDIATED p53 GENE TRANSFER ON APOPTOSIS AND RADIOSENSITIVITY OF HUMAN GASTRIC CARCINOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张珊文; 肖绍文; 吕有勇

    2003-01-01

    Objective: To evaluate the effect of adenovirus- mediated p53 gene (Adp53) on apoptosis and radiosensitivity of human gastric carcinoma cell lines. Methods: Recombinant adenovirus expressing wild-type p53 gene was transferred into four human gastric carcinoma cell lines with different p53 genetic status. p53 protein expression was detected by immunohistochemistry assay and western blot assay. Cell survival was assessed using a clonogenic assay. TUNEL assay was used in determination of apoptosis. Four human gastric carcinoma cells infected with Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flow cytometry. Results: G2/M arrest, apoptosis and inhibition of tumor cell proliferation were induced by infection at Adp53 at 100 MOI which caused high transfer rate of wild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells. The radio-enhancement ratio of Adp53 at 4Gy were 3.0 for W cell, 3.6 for M cell, 2.2 for neo cell and 2.5 for 823 cell in vitro. Conclusion: This study demonstrated that Adp53 transfer increased cellular apoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53 status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.

  4. Suppression of Ultraviolet B Exposure-Mediated Activation of NF-κB in Normal Human Keratinocytes by Resveratrol

    Directory of Open Access Journals (Sweden)

    Vaqar Mustafa Adhami

    2003-01-01

    Full Text Available Chemoprevention by naturally occurring agents is a newer dimension in the management of neoplasia, including skin cancer. Solar ultraviolet (UV radiation is the major cause of skin cancer. We recently demonstrated that resveratrol (3,5,4'-trihydroxystilbene, a polyphenolic antioxidant found in grapes and red wine, imparts protection from UVB-mediated cutaneous damages in SKH-1 hairless mice. The mechanism of action of resveratrol is not clearly understood. Here, we investigated the involvement of nuclear factor kappa B (NF-κB, which is known to play a critical role in skin biology and the development of skin cancer, as the mechanism of chemoprevention of UV damage by resveratrol. In the normal human epidermal keratinocytes, resveratrol blocked UVB-mediated (40 mJ/cm2 activation of NF-κB in a dose-dependent (5, 10, and 25μM resveratrol for 24 hours as well as time-dependent (5μ/M resveratrol for 12, 24, and 48 hours fashion. Resveratrol treatment of keratinocytes also inhibited UVB-mediated 1 phosphorylation and degradation of IκBα, and 2 activation of IKKα. We suggest that NF-κB pathway plays a critical role in the chemopreventive effects of resveratrol against the adverse effects of UV radiation including photocarcinogenesis.

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

    Science.gov (United States)

    Kokkonen, Tuomo S; Karttunen, Tuomo J

    2010-02-01

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

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

    Science.gov (United States)

    Kokkonen, Tuomo S.; Karttunen, Tuomo J.

    2010-01-01

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

  7. Cysteine amide adduct formation from carboxylic acid drugs via UGT-mediated bioactivation in human liver microsomes.

    Science.gov (United States)

    Harada, H; Toyoda, Y; Endo, T; Kobayashi, M

    2015-10-01

    Although chemical trapping has been widely used to evaluate cytochrome P450-mediated drug bioactivation, thus far, only a few in vitro-trapping studies have been performed on UDP-glucuronosyltransferase (UGT)-mediated drug bioactivation. In this study, we used cysteine (Cys) as trapping agent to gain new insights into the UGT-mediated bioactivation involving acyl glucuronides of carboxylic acid drugs. Diclofenac, ketoprofen and ibuprofen were incubated in human liver microsomes with UDPGA and Cys, followed by analysis using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The N-acyl-Cys amide adduct of diclofenac was characterized by mass analysis and was detectable even in photodiode array analysis. Our data indicated that the formation of such adducts reflects the reactivity of the corresponding acyl glucuronides. In addition, it was suggested that the adduct formation requires an N-terminal Cys moiety with both a free amine and a free thiol group, from the results using various cysteine derivatives. We propose that the S-acyl-Cys thioester adduct can form via transacylation of an acyl glucuronide and can then form to an N-acyl-Cys amide adduct through intramolecular S- to N-acyl rearrangement. This series of the reactions has important implications as a possible bioactivation mechanism for covalent binding of carboxylic acid drugs to macromolecules.

  8. Effects of Secondary Metabolites from the Fungus Septofusidium berolinense on DNA Cleavage Mediated by Human Topoisomerase IIα.

    Science.gov (United States)

    Vann, Kendra R; Ekiz, Güner; Zencir, Sevil; Bedir, Erdal; Topcu, Zeki; Osheroff, Neil

    2016-03-21

    Two metabolites from the ascomycete fungus Septofusidium berolinense were recently identified as having antineoplastic activity [Ekiz et al. (2015) J. Antibiot. , DOI: 10.1038/ja.2015.84]. However, the basis for this activity is not known. One of the compounds [3,6-dihydroxy-2-propylbenzaldehyde (GE-1)] is a hydroquinone, and the other [2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2)] is a quinone. Because some hydroquinones and quinones act as topoisomerase II poisons, the effects of GE-1 and GE-2 on DNA cleavage mediated by human topoisomerase IIα were assessed. GE-2 enhanced DNA cleavage ∼4-fold and induced scission with a site specificity similar to that of the anticancer drug etoposide. Similar to other quinone-based topoisomerase II poisons, GE-2 displayed several hallmark characteristics of covalent topoisomerase II poisons, including (1) the inability to poison a topoisomerase IIα construct that lacks the N-terminal domain, (2) the inhibition of DNA cleavage when the compound was incubated with the enzyme prior to the addition of plasmid, and (3) the loss of poisoning activity in the presence of a reducing agent. In contrast to GE-2, GE-1 did not enhance DNA cleavage mediated by topoisomerase IIα except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. These results suggest that topoisomerase IIα may play a role in mediating the cytotoxic effects of these fungal metabolites.

  9. Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair.

    Science.gov (United States)

    Tseng, Ting-Chen; Hsieh, Fu-Yu; Dai, Niann-Tzyy; Hsu, Shan-Hui

    2016-09-01

    Cell- and gene-based therapies have emerged as promising strategies for treating neurological diseases. The sources of neural stem cells are limited while the induced pluripotent stem (iPS) cells have risk of tumor formation. Here, we proposed the generation of self-renewable, multipotent, and neural lineage-related neural crest stem-like cells by chitosan substrate-mediated gene transfer of a single factor forkhead box D3 (FOXD3) for the use in neural repair. A simple, non-toxic, substrate-mediated method was applied to deliver the naked FOXD3 plasmid into human fibroblasts. The transfection of FOXD3 increased cell proliferation and up-regulated the neural crest marker genes (FOXD3, SOX2, and CD271), stemness marker genes (OCT4, NANOG, and SOX2), and neural lineage-related genes (Nestin, β-tubulin and GFAP). The expression levels of stemness marker genes and neural crest maker genes in the FOXD3-transfected fibroblasts were maintained until the fifth passage. The FOXD3 reprogrammed fibroblasts based on the new method significantly rescued the neural function of the impaired zebrafish. The chitosan substrate-mediated delivery of naked plasmid showed feasibility in reprogramming somatic cells. Particularly, the FOXD3 reprogrammed fibroblasts hold promise as an easily accessible cellular source with neural crest stem-like behavior for treating neural diseases in the future.

  10. Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells

    Science.gov (United States)

    Lin, Chaolong; Li, Huanhuan; Hao, Mengru; Xiong, Dan; Luo, Yong; Huang, Chenghao; Yuan, Quan; Zhang, Jun; Xia, Ningshao

    2016-01-01

    Genetically modified HSV-1 viruses serve as promising vectors for tumour therapy and vaccine development. The CRISPR/Cas9 system is one of the most powerful tools for precise gene editing of the genomes of organisms. However, whether the CRISPR/Cas9 system can precisely and efficiently make gene replacements in the genome of HSV-1 remains essentially unknown. Here, we reported CRISPR/Cas9-mediated editing of the HSV-1 genome in human cells, including the knockout and replacement of large genes. In established cells stably expressing CRISPR/Cas9, gRNA in coordination with Cas9 could direct a precise cleavage within a pre-defined target region, and foreign genes were successfully used to replace the target gene seamlessly by HDR-mediated gene replacement. Introducing the NHEJ inhibitor SCR7 to the CRISPR/Cas9 system greatly facilitated HDR-mediated gene replacement in the HSV-1 genome. We provided the first genetic evidence that two copies of the ICP0 gene in different locations on the same HSV-1 genome could be simultaneously modified with high efficiency and with no off-target modifications. We also developed a revolutionized isolation platform for desired recombinant viruses using single-cell sorting. Together, our work provides a significantly improved method for targeted editing of DNA viruses, which will facilitate the development of anti-cancer oncolytic viruses and vaccines. PMID:27713537

  11. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  12. The α3β4 nAChR partial agonist AT-1001 attenuates stress-induced reinstatement of nicotine seeking in a rat model of relapse and induces minimal withdrawal in dependent rats.

    Science.gov (United States)

    Yuan, Menglu; Malagon, Ariana M; Yasuda, Dennis; Belluzzi, James D; Leslie, Frances M; Zaveri, Nurulain T

    2017-08-30

    The strong reinforcing effects of nicotine and the negative symptoms such as anxiety experienced during a quit attempt often lead to relapse and low success rates for smoking cessation. Treatments that not only block the reinforcing effects of nicotine but also attenuate the motivation to relapse are needed to improve cessation rates. Recent genetic and preclinical studies have highlighted the involvement of the α3, β4, and α5 nicotinic acetylcholine receptor (nAChR) subunits and the α3β4 nAChR subtype in nicotine dependence and withdrawal. However, the involvement of these nAChR in relapse is not fully understood. We previously reported that the α3β4 nAChR partial agonist AT-1001 selectively decreases nicotine self-administration in rats without affecting food responding. In the present experiments, we examined the efficacy of AT-1001 in attenuating reinstatement of nicotine-seeking behavior in a model of stress-induced relapse. Rats extinguished from nicotine self-administration were treated with the pharmacological stressor yohimbine prior to AT-1001 treatment and reinstatement testing. We also examined whether AT-1001 produced any withdrawal-related effects when administered to nicotine-dependent rats. We found that AT-1001 dose-dependently reduced yohimbine stress-induced reinstatement of nicotine seeking. When administered to nicotine-dependent rats at the dose that significantly blocked nicotine reinstatement, AT-1001 elicited minimal somatic withdrawal signs in comparison to the nicotinic antagonist mecamylamine, which is known to produce robust withdrawal. Our data suggest that α3β4 nAChR-targeted compounds may be a promising approach for nicotine addiction treatment because they can not only block nicotine's reinforcing effects, but also decrease motivation to relapse without producing significant withdrawal effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. TALEN- and CRISPR/Cas9-Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid-Based Transfection.

    Science.gov (United States)

    Hendriks, William T; Jiang, Xin; Daheron, Laurence; Cowan, Chad A

    2015-08-03

    Using custom-engineered nuclease-mediated genome editing, such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single-stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications.

  14. Immunity and AAV-Mediated Gene Therapy for Muscular Dystrophies in Large Animal Models and Human Trials.

    Science.gov (United States)

    Wang, Zejing; Tapscott, Stephen J; Chamberlain, Jeffrey S; Storb, Rainer

    2011-01-01

    Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery specific to muscles for treating muscular dystrophies and non-muscle diseases in large animal models and human trials, factors that influence the intensity of the immune responses, and immune modulatory strategies to prevent unwanted immune responses and induce tolerance to the vector and therapeutic gene for a successful gene therapy.

  15. Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells.

    Science.gov (United States)

    Hoffmann, Kristina; Xifró, Rosa Altarcheh; Hartweg, Julia Lisa; Spitzlei, Petra; Meis, Kirsten; Molderings, Gerhard J; von Kügelgen, Ivar

    2013-01-30

    The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

  16. Social Support Mediates Loneliness and Human Herpesvirus Type 6 (HHV-6) Antibody Titers.

    Science.gov (United States)

    Dixon, Denise; Cruess, Stacy; Kilbourn, Kristin; Klimas, Nancy; Fletcher, Mary Ann; Ironson, Gail; Baum, Andrew; Schneiderman, Neil; Antoni, Michael H

    2006-07-31

    The current study investigated the impact of a severe environmental stressor and the role that declining social integration played in mediating its effect on loneliness and immune status. Increased loneliness and decreased social support in the months following the stressor (storm) were significantly associated with increased HHV-6 antibody titers, reflecting poorer control over the virus. Poorer social integration mediated the relationship between loneliness and HHV-6, even after controlling for nonspecific polyclonal B-cell activation, disease status (CD3+CD4+ cell counts), living arrangements, acute social losses (bereavement), and potential disruptions in social-support resources. These findings suggest that specific elements of social support may explain the oft-noted negative effects of loneliness on the immune system, and generalized to a medically vulnerable population.

  17. Immunotherapy: Shifting the Balance of Cell-Mediated Immunity and Suppression in Human Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Tucker, Jo A.; Jochems, Caroline [Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Gulley, James L. [Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Schlom, Jeffrey, E-mail: js141c@nih.gov; Tsang, Kwong Y. [Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2012-12-11

    Active immunotherapy is dependent on the ability of the immune system to recognize and respond to tumors. Despite overwhelming evidence to support a cell-mediated immune response to prostate cancer, it is insufficient to eradicate the disease. This is likely due to a high level of suppression at the tumor site from a variety of sources, including immunosuppressive cells. Immune cells entering the tumor microenvironment may be inhibited directly by the tumor, stromal cells or other immune cells that have been induced to adopt a suppressive phenotype. The resurgence of interest in immunotherapy following the approval of sipuleucel-T and ipilimumab by the Food and Drug Administration has brought about new strategies for overcoming tumor-mediated suppression and bolstering anti-tumor responses. Improved understanding of the immune response to prostate cancer can lead to new combination therapies, such as the use of vaccine with small molecule and checkpoint inhibitors or other immunotherapies.

  18. Immunotherapy: Shifting the Balance of Cell-Mediated Immunity and Suppression in Human Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Jeffrey Schlom

    2012-12-01

    Full Text Available Active immunotherapy is dependent on the ability of the immune system to recognize and respond to tumors. Despite overwhelming evidence to support a cell-mediated immune response to prostate cancer, it is insufficient to eradicate the disease. This is likely due to a high level of suppression at the tumor site from a variety of sources, including immunosuppressive cells. Immune cells entering the tumor microenvironment may be inhibited directly by the tumor, stromal cells or other immune cells that have been induced to adopt a suppressive phenotype. The resurgence of interest in immunotherapy following the approval of sipuleucel-T and ipilimumab by the Food and Drug Administration has brought about new strategies for overcoming tumor-mediated suppression and bolstering anti-tumor responses. Improved understanding of the immune response to prostate cancer can lead to new combination therapies, such as the use of vaccine with small molecule and checkpoint inhibitors or other immunotherapies.

  19. Fear of evaluation in social anxiety: mediation of attentional bias to human faces.

    Science.gov (United States)

    Sluis, Rachel A; Boschen, Mark J

    2014-12-01

    Social anxiety disorder (SAD) is a debilitating psychological disorder characterised by excessive fears of one or more social or performance situations, where there is potential for evaluation by others. A recently expanded cognitive-behavioural model of SAD emphasizes that both the fear of negative evaluation (FNE) and the fear of positive evaluation (FPE) contribute to enduring symptoms of SAD. Research also suggests that socially anxious individuals may show biases toward threat relevant stimuli, such as angry faces. The current study utilised a modified version of the pictorial dot-probe task in order to examine whether FNE and FPE mediate the relationship between social anxiety and an attentional bias. A group of 38 participants with moderate to high levels of self-reported social anxiety were tested in groups of two to four people and were advised that they would be required to deliver an impromptu speech. All participants then completed an assessment of attentional bias using angry-neutral, happy-neutral, and angry-happy face pairs. Conditions were satisfied for only one mediation model, indicating that the relationship between social anxiety and attentional avoidance of angry faces was mediated by FPE. These findings have important clinical implications for types of treatment concerning cognitive symptoms of SAD, along with advancing models of social anxiety. Limitations and ideas for future research from the current study were also discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

    Science.gov (United States)

    Xu, Kai; Lin, Jing; Zandi, Roza; Roth, Jack A; Ji, Lin

    2016-07-21

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

  1. Temporal dynamics of the knowledge-mediated visual disambiguation process in humans: a magnetoencephalography study.

    Science.gov (United States)

    Urakawa, Tomokazu; Ogata, Katsuya; Kimura, Takahiro; Kume, Yuko; Tobimatsu, Shozo

    2015-01-01

    Disambiguation of a noisy visual scene with prior knowledge is an indispensable task of the visual system. To adequately adapt to a dynamically changing visual environment full of noisy visual scenes, the implementation of knowledge-mediated disambiguation in the brain is imperative and essential for proceeding as fast as possible under the limited capacity of visual image processing. However, the temporal profile of the disambiguation process has not yet been fully elucidated in the brain. The present study attempted to determine how quickly knowledge-mediated disambiguation began to proceed along visual areas after the onset of a two-tone ambiguous image using magnetoencephalography with high temporal resolution. Using the predictive coding framework, we focused on activity reduction for the two-tone ambiguous image as an index of the implementation of disambiguation. Source analysis revealed that a significant activity reduction was observed in the lateral occipital area at approximately 120 ms after the onset of the ambiguous image, but not in preceding activity (about 115 ms) in the cuneus when participants perceptually disambiguated the ambiguous image with prior knowledge. These results suggested that knowledge-mediated disambiguation may be implemented as early as approximately 120 ms following an ambiguous visual scene, at least in the lateral occipital area, and provided an insight into the temporal profile of the disambiguation process of a noisy visual scene with prior knowledge.

  2. Beryllium alters lipopolysaccharide-mediated intracellular phosphorylation and cytokine release in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M; Gupta, Goutam; McCleskey, T Mark; Chaudhary, Anu

    2009-12-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide-mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1beta is enhanced. In addition, not all lipopolysaccharide-mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate-treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1beta secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling.

  3. Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal α7 from Muscle α1(2)βγδ nAChRs.

    Science.gov (United States)

    Bourne, Yves; Sulzenbacher, Gerlind; Radić, Zoran; Aráoz, Rómulo; Reynaud, Morgane; Benoit, Evelyne; Zakarian, Armen; Servent, Denis; Molgó, Jordi; Taylor, Palmer; Marchot, Pascale

    2015-06-02

    Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal α7, α4β2, α3β2, and muscle-type α12βγδ nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the α7 and α12βγδ subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 Å resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. The 3,7-diazabicyclo[3.3.1]nonane scaffold for subtype selective nicotinic acetylcholine receptor (nAChR) ligands. Part 1: the influence of different hydrogen bond acceptor systems on alkyl and (hetero)aryl substituents.

    Science.gov (United States)

    Eibl, Christoph; Tomassoli, Isabelle; Munoz, Lenka; Stokes, Clare; Papke, Roger L; Gündisch, Daniela

    2013-12-01

    3,7-Diazabicyclo[3.3.1]nonane is a naturally occurring scaffold interacting with nicotinic acetylcholine receptors (nAChRs). When one nitrogen of the 3,7-diazabicyclo[3.3.1]nonane scaffold was implemented in a carboxamide motif displaying a hydrogen bond acceptor (HBA) functionality, compounds with higher affinities and subtype selectivity for α4β2(∗) were obtained. The nature of the HBA system (carboxamide, sulfonamide, urea) had a strong impact on nAChR interaction. High affinity ligands for α4β2(∗) possessed small alkyl chains, small un-substituted hetero-aryl groups or para-substituted phenyl ring systems along with a carboxamide group. Electrophysiological responses of selected 3,7-diazabicyclo[3.3.1]nonane derivatives to Xenopus oocytes expressing various nAChR subtypes showed diverse activation profiles. Compounds with strongest agonistic profiles were obtained with small alkyl groups whereas a shift to partial agonism/antagonism was observed for aryl substituents. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Expression of a Humanized Viral 2A-Mediated lux Operon Efficiently Generates Autonomous Bioluminescence in Human Cells

    Science.gov (United States)

    Xu, Tingting; Ripp, Steven; Sayler, Gary S.; Close, Dan M.

    2014-01-01

    Background Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines. Methodology/Principal Findings The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations. Conclusions/Significance Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines

  6. Copper metabolism domain-containing 1 represses the mediators involved in the terminal effector pathways of human labour and delivery.

    Science.gov (United States)

    Lappas, Martha

    2016-04-01

    Does Copper Metabolism MURR1 Domain 1 (COMMD1) play a role in regulating the mediators involved in the terminal processes of human labour and delivery? COMMD1 plays a critical role in the termination of nuclear factor-κB (NF-κB) activity and the control of pro-inflammatory and pro-labour mediators. Inflammation and infection are the biggest aetiological factors associated with preterm birth. NF-κB drives the transcription of pro-inflammatory mediators involved in the terminal effector pathways of human labour and delivery. In non-gestational tissues, COMMD1 is a negative regulator of NF-κB-induced inflammation. The mRNA and/or protein level of COMMD1 was assessed in myometrium (n = 8 per group) and fetal membranes (n = 8 per group) obtained from term non-labouring and labouring women at term, and fetal membranes (n = 8 per group) at preterm with and without histological chorioamnionitis. Primary human myometrial cells were used to determine the effect of pro-inflammatory mediators on COMMD1 level, and the effect of COMMD1 small interfering RNA (siRNA) on pro-labour mediators. Statistical significance was ascribed to a P labour in myometrium; in fetal membranes with histologically confirmed chorioamnionitis and in myometrial cells treated with pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, the bacterial product fibroblast-stimulating lipopeptide and the viral double stranded RNA analogue polyinosinic polycytidilic acid. Loss-of-function studies revealed an increase in inflammation- and infection-induced TNF-α, IL-1α, IL-1β, IL-6, IL-8 and/or monocyte chemoattractant protein-1 mRNA abundance and/or release; and cyclo-oxygenase-2 mRNA level, release of prostaglandin (PG) F2α and mRNA level of the PGF2α receptor FP. In addition, siRNA knockdown of COMMD1 was associated with significantly increased NF-κB activation as evidenced by increased IL-1β-induced IκB-α protein degradation and NF-κB DNA binding activity. The

  7. Reinforcement as a mediator of the perception of humans by horses (Equus caballus).

    Science.gov (United States)

    Sankey, Carol; Richard-Yris, Marie-Annick; Henry, Séverine; Fureix, Carole; Nassur, Fouad; Hausberger, Martine

    2010-09-01

    A central question in the interspecific human/animal relationship is how domestic animals perceive humans as a significant element of their environment. In this study, we tested the hypothesis that the use of positive or negative reinforcement in horse training may have consequences on the animals' perception of humans, as a positive, negative or neutral element. Two groups of ponies were trained to walk backwards in response to a vocal order using either positive or negative reinforcement. Heart rate monitors and behavioural observations were used to assess the animals' perception of humans on the short (just after training) and long (5 months later) terms. The results showed that the type of reinforcement had a major effect on the subsequent animals' perception of familiar and unfamiliar humans. Negative reinforcement was rapidly associated with an increased emotional state, as revealed by heart rate measurements and behavioural observations (head movements and ears laid back position). Its use led the ponies to seek less contact with humans. On the contrary, ponies trained with positive reinforcement showed an increased interest in humans and sought contact after training. This is especially remarkable as it was reached in a maximum of 5 sessions of 1 to 3 min (i.e. 5 to 15 min) and had lasting effects (visible after 5 months). Even learning was positively influenced by positive reinforcement. Overall, horses seem capable of associating humans to particular experiences and display extended long-term memory abilities.

  8. Receptor-mediated endocytosis of α-galactosidase A in human podocytes in Fabry disease

    DEFF Research Database (Denmark)

    Prabakaran, Thaneas; Nielsen, Rikke; Larsen, Jakob Vejby;

    2011-01-01

    Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocy...

  9. Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

    DEFF Research Database (Denmark)

    Prabakaran, Thaneas; Nielsen, Rikke; Larsen, Jakob Vejby;

    2011-01-01

    Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocy...

  10. Human Papillomavirus-mediated cervical cancer awareness and Gardasil vaccination: a pilot survey among North Indian women.

    Science.gov (United States)

    Pandey, Saumya; Chandravati

    2013-10-01

    Human Papillomavirus (HPV)-mediated cervical cancer is a leading cause of morbidity and mortality in women worldwide, including Indian women. Cervical cancer control and prevention strategies are being adopted in developing nations to reduce the increasing burden of HPV infection in the vaccine era. The present study, therefore, aimed to evaluate cervical cancer awareness and knowledge of Gardasil vaccination in North Indian women. A pilot survey was conducted among 103 women of North Indian ethnicity residing in Lucknow/adjoining areas in state of Uttar Pradesh, during routine screening/clinic visits from June 2012 to December 2012. The study subjects were interviewed in either Hindi or English; subsequently the awareness of HPV-mediated cervical cancer and knowledge of Gardasil vaccination was assessed in terms of "yes", "no" and "no response". The study was approved by the Institutional Review Board. Written informed consent was taken from the participants. Overall, the response of participants (n = 103) in our single-centre survey-based pilot study was well-defined. The response regarding HPV-mediated cervical cancer awareness in terms of "yes", "no" and "no response" among the study subjects was 43.7, 44.7 and 11.6 %, respectively. Furthermore, in response to knowledge of HPV vaccine Gardasil, out of 103 subjects, 28.1 % answered "yes" while 37.9 and 34.0 % stated "no" and "no response", respectively. Our pilot survey may help in assessing knowledge of HPV-mediated cervical cancer and Gardasil vaccination awareness in women, and accordingly develop cost-effective cervical cancer control and prevention/public health counseling sessions in a clinical setting.

  11. Discoidin Domain Receptor 2 Mediates Collagen-Induced Activation of Membrane-Type 1 Matrix Metalloproteinase in Human Fibroblasts.

    Science.gov (United States)

    Majkowska, Iwona; Shitomi, Yasuyuki; Ito, Noriko; Gray, Nathanael S; Itoh, Yoshifumi

    2017-03-07

    Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity including fibroblasts and invasive cancer cell. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it upregulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation is not clearly understood. In this study we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited collagen-induced activation of proMMP-2 and upregulation of MT1-MMP at the gene and protein level. Interestingly DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signalling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells, as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without non-helical telopeptides region compared to intact collagen fibrils. Those data suggest that DDR2 is a microenvironmental sensor that regulates fibroblasts migration in collagen-rich environment.

  12. Neutrophil killing of human umbilical vein endothelial cells is oxygen radical-mediated and enhanced by TNF-. alpha

    Energy Technology Data Exchange (ETDEWEB)

    Dame, M.K.; Varani, J.; Weinberg, J.M.; Ward, P.A. (Univ. of Michigan, Ann Arbor (United States))

    1991-03-11

    Human umbilical vein endothelial cells are sensitive to killing by activated human neutrophils. Killing is inhibited in the presence of catalase and deferoxamine mesylate but not soybean trypsin inhibitor. Reagent hydrogen peroxide can substitute for activated neutrophils in producing endothelial cell injury. These data suggest that lethal injury is due to the production of oxygen radicals by activated neutrophils. In these respects, the human umbilical vein endothelial cells are similar to rat pulmonary artery endothelial cells in that pretreatment with TNF-{alpha} increases sensitivity to injury by activated neutrophils. In part, the increased endothelial cell sensitivity to killing by neutrophils may be due to up-regulation of surface adhesion molecules. However, it was observed that cells passaged more than two times in culture did not demonstrate increased killing after treatment with TNF-{alpha} while up-regulation of neutrophil adhesion could be detected through several additional passages. Although the human umbilical vein endothelial cells are qualitatively similar to rat pulmonary artery endothelial cells in their sensitivity to killing, they are quantitatively much more resistant. What accounts for the relative resistance of the human umbilical vein endothelial cells is not fully understood. In the rat pulmonary artery endothelial cells, killing is known to be dependent on an intraendothelial source of iron. Pre-treatment of the human umbilical vein endothelial cells with 8-hydroxyquinoline-bound iron increased their sensitivity to oxidant injury. These data suggest that the availability of iron within the human umbilical vein endothelial cells may be a limiting factor in sensitivity to oxygen radical-mediated injury.

  13. Killing effect of adenoviral mediated cytosine deaminase gene on human pancreatic cancer cell line PaTu 8988

    Institute of Scientific and Technical Information of China (English)

    PAN Xue; LI Zhao-shen; XU Guo-ming; CUI Long; ZHANG Su-zhen; GONG Yan-fang; TU Zhen-xing

    2001-01-01

    Objective: To evaluate the in vitro killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic carcinoma. Methods: Cytosine Deaminase (CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria, and the products containing green fluorescent protein (GFP)were propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line 8988 were infected with this virus, then 5-FC was added; XTT assay was used to estimate the relative numbers of viable cells. Results: The positive clones were confirmed by using endonuclease digestion, and the titer of the virus containing CD gene was 2 × 1011 pfu/ml. It was found that 5-FC possessed significant cytotoxic activities for CD gene transfected 8988cell line, but had little effects on non-transfected pancreatic carcinoma cells. Conclusion: CD gene mediated by adenovirus has a high infectivity and is efficient for killing cultured pancreatic carcinoma cells, indicating suicide gene may be effective for pancreatic cancer in furure.

  14. The NRF2 transcriptional target, OSGIN1, contributes to monomethyl fumarate-mediated cytoprotection in human astrocytes

    Science.gov (United States)

    Brennan, Melanie S.; Matos, Maria F.; Richter, Karl E.; Li, Bing; Scannevin, Robert H.

    2017-01-01

    Dimethyl fumarate (DMF) is indicated for the treatment of relapsing multiple sclerosis and may exert therapeutic effects via activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway. Following oral DMF administration, central nervous system (CNS) tissue is predominantly exposed to monomethyl fumarate (MMF), the bioactive metabolite of DMF, which can stabilize NRF2 and induce antioxidant gene expression; however, the detailed NRF2-dependent mechanisms modulated by MMF that lead to cytoprotection are unknown. Our data identify a mechanism for MMF-mediated cytoprotection in human astrocytes that functions in an OSGIN1-dependent manner, specifically via upregulation of the OSGIN1-61 kDa isoform. NRF2-dependent OSGIN1 expression induced P53 nuclear translocation following MMF administration, leading to cell-cycle inhibition and cell protection against oxidative challenge. This study provides mechanistic insight into MMF-mediated cytoprotection via NRF2, OSGIN1, and P53 in human CNS-derived cells and contributes to our understanding of how DMF may act clinically to ameliorate pathological processes in neurodegenerative disease. PMID:28181536

  15. Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

    Directory of Open Access Journals (Sweden)

    Kulkarni Anagha

    2009-01-01

    Full Text Available Abstract Background Human Rad51 (RAD51, analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA (dsDNA in the presence of certain nucleotide cofactors. ATP hydrolysis is not required for this process, because even ATP non hydrolysable analogs like AMP-PNP and ATPγS, support DNA unwinding. Even ADP, the product of ATP hydrolysis, feebly supports DNA unwinding. Results We find that human Rad52 (RAD52 stimulates RAD51 mediated DNA unwinding in the presence of all Adenine nucleotide cofactors, (except in AMP and no nucleotide conditions that intrinsically fail to support unwinding reaction while enhancing aggregation of RAD51-dsDNA complexes in parallel. Interestingly, salt at low concentration can substitute the role of RAD52, in facilitating aggregation of RAD51-dsDNA complexes, that concomitantly also leads to better unwinding. Conclusion RAD52 itself being a highly aggregated protein perhaps acts as scaffold to bring together RAD51 and DNA molecules into large co-aggregates of RAD52-RAD51-DNA complexes to promote RAD51 mediated DNA unwinding reaction, when appropriate nucleotide cofactors are available, presumably through macromolecular crowding effects. Our work highlights the functional link between aggregation of protein-DNA complexes and DNA unwinding in RAD51 system.

  16. Transactivation Domain of Human c-Myc Is Essential to Alleviate Poly(Q)-Mediated Neurotoxicity in Drosophila Disease Models.

    Science.gov (United States)

    Raj, Kritika; Sarkar, Surajit

    2017-03-18

    Polyglutamine (poly(Q)) disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, represent a group of neurological disorders which arise due to an atypically expanded poly(Q) tract in the coding region of the affected gene. Pathogenesis of these disorders inside the cells begins with the assembly of these mutant proteins in the form of insoluble inclusion bodies (IBs), which progressively sequester several vital cellular transcription factors and other essential proteins, and finally leads to neuronal dysfunction and apoptosis. We have shown earlier that targeted upregulation of Drosophila myc (dmyc) dominantly suppresses the poly(Q) toxicity in Drosophila. The present study examines the ability of the human c-myc proto-oncogene and also identifies the specific c-Myc isoform which drives the mitigation of poly(Q)-mediated neurotoxicity, so that it could be further substantiated as a potential drug target. We report for the first time that similar to dmyc, tissue-specific induced expression of human c-myc also suppresses poly(Q)-mediated neurotoxicity by an analogous mechanism. Among the three isoforms of c-Myc, the rescue potential was maximally manifested by the full-length c-Myc2 protein, followed by c-Myc1, but not by c-MycS which lacks the transactivation domain. Our study suggests that strategies focussing on the transactivation domain of c-Myc could be a very useful approach to design novel drug molecules against poly(Q) disorders.

  17. Expression of human clotting factor Ⅸ mediated by recombinant lentiviral vector in cultured cells and hemophilia B mice

    Institute of Scientific and Technical Information of China (English)

    ZHU Huanzhang; CHEN Xiaoguang; LI Feng; GONG Juli; XUE Jinglun

    2003-01-01

    To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMVΔR8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.

  18. Retracted: Physiologically based pharmacokinetic predictions of intestinal BCRP-mediated effect of telmisartan on the pharmacokinetics of rosuvastatin in humans.

    Science.gov (United States)

    Bae, Soo Hyeon; Park, Wan-Su; Han, Seunghoon; Park, Gab-Jin; Lee, Jongtae; Hong, Taegon; Jeon, Sangil; Yim, Dong-Seok

    2017-07-01

    'Physiologically based pharmacokinetic predictions of intestinal BCRP-mediated effect of telmisartan on the pharmacokinetics of rosuvastatin in humans' by Soo Hyeon Bae, Wan-Su Park, Seunghoon Han, Gab-jin Park, Jongtae Lee, Taegon Hong, Sangil Jeon and Dong-Seok Yim The above article, published online on 06 February 2017 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor in Chief, K. Sandy Pang, and John Wiley & Sons, Ltd. The authors retracted the paper due to errors associated with use of log D vs. log P of telmisartan as inputs of the PBPK model. The authors concluded that there are too many changes in the article to be resolved by an Erratum, and had requested a retraction. Reference Bae, S. H., Park, W.-S., Han, S., Park, G., Lee, J., Hong, T., Jeon, S., and Yim, D.-S. (2016) Physiologically based pharmacokinetic predictions of intestinal BCRP-mediated effect of telmisartan on the pharmacokinetics of rosuvastatin in humans. Biopharm. Drug Dispos., doi: 10.1002/bdd.2060. Copyright © 2016 John Wiley & Sons, Ltd.

  19. α7 Nicotinic receptor-mediated astrocytic gliotransmitter release: Aβ effects in a preclinical Alzheimer's mouse model.

    Directory of Open Access Journals (Sweden)

    Tiina Maria Pirttimaki

    Full Text Available It is now recognized that astrocytes participate in synaptic communication through intimate interactions with neurons. A principal mechanism is through the release of gliotransmitters (GTs such as ATP, D-serine and most notably, glutamate, in response to astrocytic calcium elevations. We and others have shown that amyloid-β (Aβ, the toxic trigger for Alzheimer's disease (AD, interacts with hippocampal α7 nicotinic acetylcholine receptors (nAChRs. Since α7nAChRs are highly permeable to calcium and are expressed on hippocampal astrocytes, we investigated whether Aβ could activate astrocytic α7nAChRs in hippocampal slices and induce GT glutamate release. We found that biologically-relevant concentrations of Aβ1-42 elicited α7nAChR-dependent calcium elevations in hippocampal CA1 astrocytes and induced NMDAR-mediated slow inward currents (SICs in CA1 neurons. In the Tg2576 AD mouse model for Aβ over-production and accumulation, we found that spontaneous astrocytic calcium elevations were of higher frequency compared to wildtype (WT. The frequency and kinetic parameters of AD mice SICs indicated enhanced gliotransmission, possibly due to increased endogenous Aβ observed in this model. Activation of α7nAChRs on WT astrocytes increased spontaneous inward currents on pyramidal neurons while α7nAChRs on astrocytes of AD mice were abrogated. These findings suggest that, at an age that far precedes the emergence of cognitive deficits and plaque deposition, this mouse model for AD-like amyloidosis exhibits augmented astrocytic activity and glutamate GT release suggesting possible repercussions for preclinical AD hippocampal neural networks that contribute to subsequent cognitive decline.

  20. Positive inotropy mediated via CGRP receptors in isolated human myocardial trabeculae

    DEFF Research Database (Denmark)

    Saetrum Opgaard, O; Hasbak, P; de Vries, R;

    2000-01-01

    Isometric contractile force were studied on isolated human myocardial trabeculae that were paced at 1.0 Hz in tissue baths. Alpha calcitonin gene-related peptide (alpha-CGRP) had a potent positive inotropic effect in most trabeculae from both the right atrium and left ventricle, and this effect...... reaction (PCR) mRNAs encoding the human calcitonin receptor-like receptor and the receptor associated modifying proteins (RAMPs) RAMP1, RAMP2, and RAMP3 were detected in human myocardial trabeculae from both the right atrium and left ventricle. In conclusion, functional CGRP(1) and CGRP(2) receptors may...

  1. Human CYP2E1 mediates the formation of glycidamide from acrylamide.

    Science.gov (United States)

    Settels, Eva; Bernauer, Ulrike; Palavinskas, Richard; Klaffke, Horst S; Gundert-Remy, Ursula; Appel, Klaus E

    2008-10-01

    Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species.

  2. Human CYP2E1 mediates the formation of glycidamide from acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Settels, Eva; Appel, Klaus E. [Federal Institute for Risk Assessment, Center for Experimental Toxicology, Berlin (Germany); Bernauer, Ulrike; Gundert-Remy, Ursula [Federal Institute for Risk Assessment, Department of Safety of Substances and Preparations, Berlin (Germany); Palavinskas, Richard; Klaffke, Horst S. [Federal Institute for Risk Assessment, Center for Analytical Chemistry, Berlin (Germany)

    2008-10-15

    Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes trademark, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes trademark, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes trademark; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species. (orig.)

  3. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

    OpenAIRE

    Kontny, U.; Kurane, I; Ennis, F A

    1988-01-01

    It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

  4. Timberol® Inhibits TAAR5-Mediated Responses to Trimethylamine and Influences the Olfactory Threshold in Humans.

    Directory of Open Access Journals (Sweden)

    Ivonne Wallrabenstein

    Full Text Available In mice, trace amine-associated receptors (TAARs are interspersed in the olfactory epithelium and constitute a chemosensory subsystem that is highly specific for detecting volatile amines. Humans possess six putative functional TAAR genes. Human TAAR5 (hTAAR5 is highly expressed in the olfactory mucosa and was shown to be specifically activated by trimethylamine. In this study, we were challenged to uncover an effective blocker substance for trimethylamine-induced hTAAR5 activation. To monitor blocking effects, we recombinantly expressed hTAAR5 and employed a commonly used Cre-luciferase reporter gene assay. Among all tested potential blocker substances, Timberol®, an amber-woody fragrance, is able to inhibit the trimethylamine-induced hTAAR5 activation up to 96%. Moreover, human psychophysical data showed that the presence of Timberol® increases the olfactory detection threshold for the characteristic fishy odor of trimethylamine by almost one order of magnitude. In conclusion, our results show that among tested receptors Timberol® is a specific and potent antagonist for the hTAAR5-mediated response to trimethylamine in a heterologous system. Furthermore, our data concerning the observed shift of the olfactory detection threshold in vivo implicate that hTAAR5 or other receptors that may be inhibited by Timberol® could be involved in the high affinity olfactory perception of trimethylamine in humans.

  5. Protease-mediated release of chemotherapeutics from mesoporous silica nanoparticles to ex vivo human and mouse lung tumors.

    Science.gov (United States)

    van Rijt, Sabine H; Bölükbas, Deniz A; Argyo, Christian; Datz, Stefan; Lindner, Michael; Eickelberg, Oliver; Königshoff, Melanie; Bein, Thomas; Meiners, Silke

    2015-03-24

    Nanoparticles allow for controlled and targeted drug delivery to diseased tissues and therefore bypass systemic side effects. Spatiotemporal control of drug release can be achieved by nanocarriers that respond to elevated levels of disease-specific enzymes. For example, matrix metalloproteinase 9 (MMP9) is overexpressed in tumors, is known to enhance the metastatic potency of malignant cells, and has been associated with poor prognosis of lung cancer. Here, we report the synthesis of mesoporous silica nanoparticles (MSNs) tightly capped by avidin molecules via MMP9 sequence-specific linkers to allow for site-selective drug delivery in high-expressing MMP9 tumor areas. We provide proof-of-concept evidence for successful MMP9-triggered drug release from MSNs in human tumor cells and in mouse and human lung tumors using the novel technology of ex vivo 3D lung tissue cultures. This technique allows for translational testing of drug delivery strategies in diseased mouse and human tissue. Using this method we show MMP9-mediated release of cisplatin, which induced apoptotic cell death only in lung tumor regions of Kras mutant mice, without causing toxicity in tumor-free areas or in healthy mice. The MMP9-responsive nanoparticles also allowed for effective combinatorial drug delivery of cisplatin and proteasome inhibitor bortezomib, which had a synergistic effect on the (therapeutic) efficiency. Importantly, we demonstrate the feasibility of MMP9-controlled drug release in human lung tumors.

  6. Notch activation is dispensable for D, L-sulforaphane-mediated inhibition of human prostate cancer cell migration.

    Directory of Open Access Journals (Sweden)

    Eun-Ryeong Hahm

    Full Text Available D, L-Sulforaphane (SFN, a synthetic racemic analog of broccoli constituent L-sulforaphane, is a highly promising cancer chemopreventive agent with in vivo efficacy against chemically-induced as well as oncogene-driven cancer in preclinical rodent models. Cancer chemopreventive effect of SFN is characterized by G(2/M phase cell cycle arrest, apoptosis induction, and inhibition of cell migration and invasion. Moreover, SFN inhibits multiple oncogenic signaling pathways often hyperactive in human cancers, including nuclear factor-κB, Akt, signal transducer and activator of transcription 3, and androgen receptor. The present study was designed to determine the role of Notch signaling, which is constitutively active in many human cancers, in anticancer effects of SFN using prostate cancer cells as a model. Exposure of human prostate cancer cells (PC-3, LNCaP, and/or LNCaP-C4-2B to SFN as well as its naturally-occurring thio-, sulfinyl-, and sulfonyl-analogs resulted in cleavage (activation of Notch1, Notch2, and Notch4, which was accompanied by a decrease in levels of full-length Notch forms especially at the 16- and 24-hour time points. The SFN-mediated cleavage of Notch isoforms was associated with its transcriptional activation as evidenced by RBP-Jk-, HES-1A/B- and HEY-1 luciferase reporter assays. Migration of PC-3 and LNCaP cells was decreased significantly by RNA interference of Notch1 and Notch2, but not Notch4. Furthermore, SFN-mediated inhibition of PC-3 and LNCaP cell migration was only marginally affected by knockdown of Notch1 and Notch2. Strikingly, SFN administration to Transgenic Adenocarcinoma of Mouse Prostate transgenic mice failed to increase levels of cleaved Notch1, cleaved Notch2, and HES-1 proteins in vivo in prostatic intraepithelial neoplasia, well-differentiated carcinoma or poorly-differentiated prostate cancer lesions. These results indicate that Notch activation is largely dispensable for SFN-mediated inhibition of cell

  7. Dehydroeffusol effectively inhibits human gastric cancer cell-mediated vasculogenic mimicry with low toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Wenming; Meng, Mei; Zhang, Bin; Du, Longsheng; Pan, Yanyan; Yang, Ping; Gu, Zhenlun; Zhou, Quansheng, E-mail: quanshengzhou@yahoo.com; Cao, Zhifei, E-mail: hunancao@163.com

    2015-09-01

    Accumulated data has shown that various vasculogenic tumor cells, including gastric cancer cells, are able to directly form tumor blood vessels via vasculogenic mimicry, supplying oxygen and nutrients to tumors, and facilitating progression and metastasis of malignant tumors. Therefore, tumor vasculogenic mimicry is a rational target for developing novel anticancer therapeutics. However, effective antitumor vasculogenic mimicry-targeting drugs are not clinically available. In this study, we purified 2,7-dihydroxyl-1-methyl-5-vinyl-phenanthrene, termed dehydroeffusol, from the traditional Chinese medicinal herb Juncus effusus L., and found that dehydroeffusol effectively inhibited gastric cancer cell-mediated vasculogenic mimicry in vitro and in vivo with very low toxicity. Dehydroeffusol significantly suppressed gastric cancer cell adhesion, migration, and invasion. Molecular mechanistic studies revealed that dehydroeffusol markedly inhibited the expression of a vasculogenic mimicry master gene VE-cadherin and reduced adherent protein exposure on the cell surface by inhibiting gene promoter activity. In addition, dehydroeffusol significantly decreased the expression of a key vasculogenic gene matrix metalloproteinase 2 (MMP2) in gastric cancer cells, and diminished MMP2 protease activity. Together, our results showed that dehydroeffusol effectively inhibited gastric cancer cell-mediated vasculogenic mimicry with very low toxicity, suggesting that dehydroeffusol is a potential drug candidate for anti-gastric cancer neovascularization and anti-gastric cancer therapy. - Highlights: • Dehydroeffusol markedly inhibits gastric cancer cell-mediated vasculogenic mimicry. • Dehydroeffusol suppresses the expression of vasculogenic mimicry key gene VE-cadherin. • Dehydroeffusol decreases the MMP2 expression and activity in gastric cancer cells. • Dehydroeffusol is a potential anti-cancer drug candidate with very low toxicity.

  8. Glutamine deprivation sensitizes human breast cancer MDA-MB-231 cells to TRIAL-mediated apoptosis.

    Science.gov (United States)

    Dilshara, Matharage Gayani; Jeong, Jin-Woo; Prasad Tharanga Jayasooriya, Rajapaksha Gedara; Neelaka Molagoda, Ilandarage Menu; Lee, Seungheon; Park, Sang Rul; Choi, Yung Hyun; Kim, Gi-Young

    2017-02-13

    Tumor cell metabolism is a promising target for various cancer treatments. Apart from aerobic glycolysis, cancer cell growth is dependent on glutamine (Gln) supply, leading to their survival and differentiation. Therefore, we examined whether treatment with TNF-related apoptosis-inducing ligand (TRAIL) sensitizes MDA-MB-231 cells to apoptosis under Gln deprivation condition (TRAIL/Gln deprivation). Gln deprivation decreased cell proliferation as expected, but did not induce remarkable cell death. TRAIL/Gln deprivation, however, significantly increased growth inhibition and morphological shrinkage of MDA-MB-231 cells compared to those induced by treatment with either Gln deprivation or TRAIL alone. Moreover, TRAIL/Gln deprivation upregulated the apoptotic sub-G1 phase accompanied with a remarkable decrease of pro-caspase-3, pro-caspase-9, and anti-apoptotic xIAP, and Bcl-2. Increased cleavage of PARP and pro-apoptotic Bid protein expression suggests that TRAIL/Gln deprivation triggers mitochondrion-mediated apoptosis in MDA-MB-231 cells. Additionally, TRAIL/Gln deprivation upregulated the expression of endoplasmic reticulum (ER) stress markers such as ATF4 and phosphorylated eIF2α, thereby enhancing the C/EBP homologous protein (CHOP) protein level. Transient knockdown of CHOP partically reversed TRAIL/Gln deprivation-mediated apoptosis. Accordingly, TRAIL/Gln deprivation enhanced the expression of death receptor 5 (DR5) and transient knockdown of DR5 completely restored TRAIL/Gln deprivation-mediated apoptosis. Taken together, our results suggest that Gln deprivation conditions can be used for the development of new therapies for TRAIL-resistant cancers.

  9. Human Factors Principles in Design of Computer-Mediated Visualization for Robot Missions

    Energy Technology Data Exchange (ETDEWEB)

    David I Gertman; David J Bruemmer

    2008-12-01

    With increased use of robots as a resource in missions supporting countermine, improvised explosive devices (IEDs), and chemical, biological, radiological nuclear and conventional explosives (CBRNE), fully understanding the best means by which to complement the human operator’s underlying perceptual and cognitive processes could not be more important. Consistent with control and display integration practices in many other high technology computer-supported applications, current robotic design practices rely highly upon static guidelines and design heuristics that reflect the expertise and experience of the individual designer. In order to use what we know about human factors (HF) to drive human robot interaction (HRI) design, this paper reviews underlying human perception and cognition principles and shows how they were applied to a threat detection domain.

  10. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells

    National Research Council Canada - National Science Library

    Tchasovnikarova, I. A; Timms, R. T; Matheson, N. J; Wals, K; Antrobus, R; Gottgens, B; Dougan, G; Dawson, M. A; Lehner, P. J

    2015-01-01

    ... (see the Perspective by Brummelkamp). They identified a complex of proteins in human cells they called HUSH that kept particular parts of the genome silent by changing associated histone methylation marks...

  11. NF-κB Mediated Transcription in Human Monocytic Cells and Endothelial Cells.

    Science.gov (United States)

    Parry, G C; Mackman, N

    1998-04-01

    Monocytes and endothelial cells become activated at sites of inflammation and contribute to the pathology of many diseases, including septic shock and atherosclerosis. In these cells, induction of genes expressing various inflammatory mediators, such as adhesion molecules, cytokines, and growth factors, is regulated by NF-κB/Rel transcription factors. Recent studies have identified components of the signal transduction pathways leading to the activation of NF-κB/Rel proteins. Inhibition of these signaling pathways provides a novel therapeutic approach to prevent inducible gene expression in both monocytes and endothelial cells. (Trends Cardiovasc Med 1998;8:138-142). © 1998, Elsevier Science Inc.

  12. Inhibition of lipid mediator biosynthesis in human inflammatory cells by BIRM 270.

    Science.gov (United States)

    Parks, T P; Hoffman, A F; Homon, C A; Graham, A G; Lazer, E S; Chilton, F H; Borgeat, P; Raible, D; Schulman, E; Bass, D A

    1995-01-01

    BIRM 270 was developed as a potent and enantioselective inhibitor of LTB4 biosynthesis by human neutrophils, and was also found to inhibit LTC4 production by human eosinophils and lung mast cells. BIRM 270 inhibited LTB4 synthesis in neutrophils by preventing arachidonate release from membrane phospholipids, and over the same concentration range, inhibited PAF biosynthesis. BIRM 270 did not directly inhibit acylhydrolases which have been implicated in eicosanoid and PAF biosynthesis, suggesting an indirect mode of action.

  13. Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor.

    OpenAIRE

    Francki, A.; Uciechowski, P.; Floege, J; von der Ohe, J.; Resch, K.; Radeke, H. H.

    1995-01-01

    For various forms of human glomerulonephritis a close relationship between inflammatory injury and a local mesangial proliferative response has been described. Herein, we used primary cultures of human glomerular mesangial cells (HMCs) from five different donors to determine the autocrine growth-inducing capacity of their supernatants after stimulation with different cytokines and lipopolysaccharide (LPS) to determine whether this effect is due to basic fibroblast growth factor (bFGF). The ba...

  14. Programmed Fetal Membrane Senescence and Exosome-Mediated Signaling: A Mechanism Associated With Timing of Human Parturition

    Directory of Open Access Journals (Sweden)

    Ramkumar Menon

    2017-08-01

    Full Text Available Human parturition is an inflammatory process that involves both fetal and maternal compartments. The precise immune cell interactions have not been well delineated in human uterine tissues during parturition, but insights into human labor initiation have been informed by studies in animal models. Unfortunately, the timing of parturition relative to fetal maturation varies among viviparous species—indicative of different phylogenetic clocks and alarms—but what is clear is that important common pathways must converge to control the birth process. Herein, we hypothesize a novel signaling mechanism initiated by human fetal membrane aging and senescence-associated inflammation. Programmed events of fetal membrane aging coincide with fetal growth and organ maturation. Mechanistically, senescence involves in telomere shortening and activation of p38 mitogen-activated signaling kinase resulting in aging-associated phenotypic transition. Senescent tissues release inflammatory signals that are propagated via exosomes to cause functional changes in maternal uterine tissues. In vitro, oxidative stress causes increased release of inflammatory mediators (senescence-associated secretory phenotype and damage-associated molecular pattern markers that can be packaged inside the exosomes. These exosomes traverse through tissues layers, reach maternal tissues to increase overall inflammatory load transitioning them from a quiescent to active state. Animal model studies have shown that fetal exosomes can travel from fetal to the maternal side. Thus, aging fetal membranes and membrane-derived exosomes cargo fetal signals to the uterus and cervix and may trigger parturition. This review highlights a novel hypothesis in human parturition research based on data from ongoing research using human fetal membrane model system.

  15. Neurally mediated airway constriction in human and other species: a comparative study using precision-cut lung slices (PCLS.

    Directory of Open Access Journals (Sweden)

    Marco Schlepütz

    Full Text Available The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS respond to electric field stimulation (EFS and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM caused bronchoconstriction in human (4 from 7 and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC. However, this effect was notably smaller in human responder (30 ± 7.1% than in guinea pig (79 ± 5.1% PCLS. The transient receptor potential (TRP channel blockers SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic