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Sample records for selective lysosomal targeting

  1. Selective tracking of lysosomal Cu2+ ions using simultaneous target- and location-activated fluorescent nanoprobes.

    PubMed

    Li, Yinhui; Zhao, Yirong; Chan, Winghong; Wang, Yijun; You, Qihua; Liu, Changhui; Zheng, Jing; Li, Jishan; Yang, Sheng; Yang, Ronghua

    2015-01-01

    Levels of lysosomal copper are tightly regulated in the human body. However, few methods for monitoring dynamic changes in copper pools are available, thus limiting the ability to diagnostically assess the influence of copper accumulation on health status. We herein report the development of a dual target and location-activated rhodamine-spiropyran probe, termed Rhod-SP, activated by the presence of lysosomal Cu(2+). Rhod-SP contains a proton recognition unit of spiropyran, which provides molecular switching capability, and a latent rhodamine fluorophore for signal transduction. Upon activation by lysosomal acidic pH, Rhod-SP binds with Cu(2+) by spiropyran-based proton activation, promoting, in turn, rhodamine ring opening, which shows a "switched on" fluorescence signal. However, to protect Rhod-SP from degradation and interference by the physiological environment, it is engineered on mesoporous silica nanoparticles (MSNs), and the surface of Rhod-SP@MSNs is further anchored with β-cyclodextrin (β-CD) to enhance the solubility and bioavailability of Rhod-SP@MSN-CD. Next, to enhance cell specificity, a guiding unit of c(RGDyK) peptide conjugated adamantane (Ad-RGD) as prototypical system, is incorporated on the surface of Rhod-SP@MSN-CD to target integrin αvβ3 and αvβ5 overexpressed on cancer cells. Fluorescence imaging showed that both Rhod-SP@MSN-CD and Rhod-SP@MSN-CD-RGD were suitable for visualizing exogenous and endogenous Cu(2+) in lysosomes of living cells. This strategy addresses some common challenges of chemical probes in biosensing, such as spatial resolution in cell imaging, the solubility and stability in biological system, and the interference from intracellular species. The newly designed nanoprobe, which allows one to track, on a location-specific basis, and visualize lysosomal Cu(2+), offers a potentially rich opportunity to examine copper physiology in both healthy and diseased states. PMID:25435382

  2. Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

    PubMed Central

    Sukhai, Mahadeo A.; Prabha, Swayam; Hurren, Rose; Rutledge, Angela C.; Lee, Anna Y.; Sriskanthadevan, Shrivani; Sun, Hong; Wang, Xiaoming; Skrtic, Marko; Seneviratne, Ayesh; Cusimano, Maria; Jhas, Bozhena; Gronda, Marcela; MacLean, Neil; Cho, Eunice E.; Spagnuolo, Paul A.; Sharmeen, Sumaiya; Gebbia, Marinella; Urbanus, Malene; Eppert, Kolja; Dissanayake, Dilan; Jonet, Alexia; Dassonville-Klimpt, Alexandra; Li, Xiaoming; Datti, Alessandro; Ohashi, Pamela S.; Wrana, Jeff; Rogers, Ian; Sonnet, Pascal; Ellis, William Y.; Corey, Seth J.; Eaves, Connie; Minden, Mark D.; Wang, Jean C.Y.; Dick, John E.; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D.

    2012-01-01

    Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML. PMID:23202731

  3. Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells.

    PubMed

    Bernard, Dannie; Gebbia, Marinella; Prabha, Swayam; Gronda, Marcela; MacLean, Neil; Wang, Xiaoming; Hurren, Rose; Sukhai, Mahadeo A; Cho, Eunice E; Manolson, Morris F; Datti, Alessandro; Wrana, Jeffrey; Minden, Mark D; Al-Awar, Rima; Aman, Ahmed; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D

    2015-07-01

    To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function. PMID:25832785

  4. Lysosomal Targeting of Cystinosin Requires AP-3.

    PubMed

    Andrzejewska, Zuzanna; Névo, Nathalie; Thomas, Lucie; Bailleux, Anne; Chauvet, Véronique; Benmerah, Alexandre; Antignac, Corinne

    2015-07-01

    Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine-based signal (GYDQL) in its C-terminal tail and a non-classical motif in its fifth inter-TM loop. Using the yeast two-hybrid system, we showed that the GYDQL motif specifically interacted with the μ subunit of the adaptor protein complex 3 (AP-3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP-3-depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP-3 knockdown cells where it also accumulated in the trans-Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63-GYDQL chimeric protein were not increased when clathrin-mediated endocytosis was impaired, our data show that the tyrosine-based motif of cystinosin is a 'strong' AP-3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway. PMID:25753619

  5. Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells.

    PubMed

    Rowson-Hodel, Ashley R; Berg, Anastasia L; Wald, Jessica H; Hatakeyama, Jason; VanderVorst, Kacey; Curiel, Daniel A; Leon, Leonardo J; Sweeney, Colleen; Carraway, Kermit L

    2016-05-28

    Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the amiloride derivative 5-(N,N-hexamethylene) amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis. PMID:26944316

  6. Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

    PubMed

    Markmann, Sandra; Thelen, Melanie; Cornils, Kerstin; Schweizer, Michaela; Brocke-Ahmadinejad, Nahal; Willnow, Thomas; Heeren, Joerg; Gieselmann, Volkmar; Braulke, Thomas; Kollmann, Katrin

    2015-07-01

    Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes. PMID:25786328

  7. Induced oligomerization targets Golgi proteins for degradation in lysosomes

    PubMed Central

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D.

    2015-01-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130’s cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  8. Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aβ Generation and Amyloid Plaque Pathogenesis

    PubMed Central

    Xiao, Qingli; Yan, Ping; Ma, Xiucui; Liu, Haiyan; Perez, Ronaldo; Zhu, Alec; Gonzales, Ernesto; Tripoli, Danielle L.; Czerniewski, Leah; Ballabio, Andrea; Cirrito, John R.

    2015-01-01

    In AD, an imbalance between Aβ production and removal drives elevated brain Aβ levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via α-cleavage at the plasma membrane, amyloidogenic β- and γ-cleavage within endosomes to generate Aβ, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aβ levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adeno-associated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the α and β C-terminal APP fragments (CTFs), and in the steady-state Aβ levels in the brain interstitial fluid. In aged mice, total Aβ levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and β-CTFs, and attenuated Aβ generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aβ generation. Activation of TFEB in neurons is an effective strategy to attenuate Aβ generation and attenuate amyloid plaque deposition in AD. SIGNIFICANCE STATEMENT A key driver for AD pathogenesis is the net balance between production and clearance of Aβ, the major component of amyloid plaques. Here we demonstrate that lysosomal degradation of holo-APP influences Aβ production by limiting the availability of APP for amyloidogenic processing. Using viral gene transfer of transcription factor EB (TFEB), a master regulator of lysosome biogenesis in neurons of APP/PS1 mice, steady-state levels of APP were reduced, resulting in decreased interstitial fluid Aβ levels and attenuated amyloid deposits. These effects were caused by accelerated lysosomal degradation of endocytosed APP, reflected by reduced APP half-life and steady-state levels in TFEB-expressing cells, with resultant decrease in Aβ production and release. Additional studies are needed to explore the therapeutic potential of this approach. PMID:26338325

  9. Protein Networks Supporting AP-3 Function in Targeting Lysosomal Membrane Proteins

    PubMed Central

    Baust, Thorsten; Anitei, Mihaela; Czupalla, Cornelia; Parshyna, Iryna; Bourel, Line; Thiele, Christoph; Krause, Eberhard

    2008-01-01

    The AP-3 adaptor complex targets selected transmembrane proteins to lysosomes and lysosome-related organelles. We reconstituted its preferred interaction with liposomes containing the ADP ribosylation factor (ARF)-1 guanosine triphosphatase (GTPase), specific cargo tails, and phosphatidylinositol-3 phosphate, and then we performed a proteomic screen to identify new proteins supporting its sorting function. We identified ?30 proteins belonging to three networks regulating either AP-3 coat assembly or septin polymerization or Rab7-dependent lysosomal transport. RNA interference shows that, among these proteins, the ARF-1 exchange factor brefeldin A-inhibited exchange factor 1, the ARF-1 GTPase-activating protein 1, the Cdc42-interacting Cdc42 effector protein 4, an effector of septin-polymerizing GTPases, and the phosphatidylinositol-3 kinase IIIC3 are key components regulating the targeting of lysosomal membrane proteins to lysosomes in vivo. This analysis reveals that these proteins, together with AP-3, play an essential role in protein sorting at early endosomes, thereby regulating the integrity of these organelles. PMID:18287518

  10. Chaperone-mediated autophagy: Dice's 'wild' idea about lysosomal selectivity.

    PubMed

    Cuervo, Ana Maria

    2011-08-01

    A little over 1 year ago, we lost a bright scientist and a dear colleague who, in his younger years, proposed the 'heretical' idea that lysosomes could selectively degrade cytosolic proteins. That scientist was J. Fred Dice, and his lifetime's discovery was the degradative pathway that we now know as chaperone-mediated autophagy. PMID:21750569

  11. Rapid biogenesis and sensitization of secretory lysosomes in NK cells mediated by target-cell recognition

    PubMed Central

    Liu, Dongfang; Xu, Liang; Yang, Fan; Li, Dongdong; Gong, Feili; Xu, Tao

    2005-01-01

    Natural killer (NK) cells play important roles in defense against tumor, viral infection, and cell-mediated xenograft rejection through secretion of secretory lysosomes. In this study, we used high time-resolution membrane capacitance measurement and fluorescence-imaging techniques to study the biogenesis and exocytosis of secretory lysosomes in a human NK cell line. We demonstrated a high-affinity Ca2+-dependent exocytosis of secretory lysosomes, which is sensitized further after target-cell stimulation. Our data also suggest an unusual rapid and dramatic de novo formation of secretory lysosomes after target-cell recognition. The rapid biogenesis of secretory lysosomes was blocked by specific protein kinase C inhibitor but not by brefeldin A. We propose that target-cell recognition triggers rapid biogenesis and sensitization of secretory lysosomes in NK cells through activation of PKC. PMID:15618404

  12. Tumor cell specific and lysosome-targeted delivery of nitric oxide for enhanced photodynamic therapy triggered by 808 nm near-infrared light.

    PubMed

    Xiang, Hui-Jing; Deng, Qiao; An, Lu; Guo, Min; Yang, Shi-Ping; Liu, Jin-Gang

    2015-12-15

    A novel cancer cell lysosome-targetable multifunctional NO-delivery nanoplatform (Lyso-Ru-NO@FA@C-TiO2) () was developed. It selectively targets folate-receptor overexpressed cancer cells and specifically locates within the lysosome organelle to which NO and reactive oxygen species are simultaneously released upon 808 nm NIR light irradiation. The dual-targeted nanoplatform () demonstrated the highest anticancer efficacy compared with nontargeted counterparts under NIR light sensitization. PMID:26503188

  13. Utilization of the indirect lysosome targeting pathway by lysosome-associated membrane proteins (LAMPs) is influenced largely by the C-terminal residue of their GYXXphi targeting signals.

    PubMed

    Gough, N R; Zweifel, M E; Martinez-Augustin, O; Aguilar, R C; Bonifacino, J S; Fambrough, D M

    1999-12-01

    A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the mu2 and mu3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively. PMID:10564644

  14. Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes

    PubMed Central

    Flannery, Andrew R.; Czibener, Cecilia

    2010-01-01

    Syt VII is a Ca2+ sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex. In macrophages, Syt VII is translocated simultaneously with the lysosomal tetraspanin CD63 from tubular lysosomes to nascent phagosomes in a Ca2+-dependent process that facilitates particle uptake. Mutations in Syt VII palmitoylation sites block trafficking of Syt VII, but not CD63, to lysosomes and phagosomes, whereas tyrosine replacement in the lysosomal targeting motif of CD63 causes both proteins to accumulate on the plasma membrane. Complexes of CD63 and Syt VII are detected only when Syt VII palmitoylation sites are intact. These findings identify palmitoylation-dependent association with the tetraspanin CD63 as the mechanism by which Syt VII is targeted to lysosomes. PMID:21041449

  15. A Lysosome-Targeting AIEgen for Autophagy Visualization.

    PubMed

    Leung, Chris Wai Tung; Wang, Zhiming; Zhao, Engui; Hong, Yuning; Chen, Sijie; Kwok, Ryan Tsz Kin; Leung, Anakin Chun Sing; Wen, Rongsen; Li, Bingshi; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2016-02-01

    In this work, a morpholine-functionalized aggregation-induced emission luminogen (AIEgen), AIE-LysoY, is reported for lysosomal imaging and autophagy visualization. To attain outstanding imaging contrast, AIE-LysoY is equipped with excited state intramolecular proton transfer (ESIPT) characteristic. AIE-LysoY provides a new platform for lysosome visualization with good biocompatibility, large Stokes shift, superior signal-to-noise ratio, and high photostability. PMID:26688031

  16. LYSOSOMAL TARGETING AND TRAFFICKING OF ACID SPHINGOMYELINASE TO LIPID RAFT PLATFORMS IN CORONARY ENDOTHELIAL CELLS

    PubMed Central

    Jin, Si; Yi, Fan; Zhang, Fan; Poklis, Justin L.; Li, Pin-Lan

    2009-01-01

    Objective To determine whether lysosome trafficking and targeting of acid sphingomyelinase (ASMase) to this organelle contribute to the formation of lipid raft (LR) signaling platforms in the membrane of coronary arterial endothelial cells (CAECs). Methods and Results By measurement of fluorescent resonance energy transfer (FRET), it was found that in FasL-stimulated CAECs, membrane lamp1 (a lysosome marker protein) or Fas and GM1 (a LR marker) were trafficking together. Cofocal colocalization assay showed that ceramide was enriched in these LR platforms. Further studies demonstrated that these ceramide molecules in LR platforms were colocalized with ASMase, a ceramide producing enzyme. Fluorescence imaging of living CAECs loaded with lysosomal specific dyes demonstrated that lysosomes fused with membrane upon FasL stimulation. In the presence of lysosome function inhibitors, bafilomycin (Baf) or glycyl-L-phenylalanine-?-naphthylamide (GPN), these FasL-induced changes were abolished. Moreover, this FasL-induced formation of LR platforms was also blocked in ECs transfected with siRNA of sortilin, an intracellular transporter for targeting of ASMase to lysosomes. Functionally, FasL-induced impairment of vasodilator response was reversed by lysosomal inhibitors or sortilin gene silencing. Conclusions Lysosomal trafficking and targeting of ASMase are importantly involved in LRs clustering in ECs membrane, leading to the formation of signaling platforms or signalosomes. PMID:18772496

  17. Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes.

    PubMed

    Moody, Paul R; Sayers, Edward J; Magnusson, Johannes P; Alexander, Cameron; Borri, Paola; Watson, Peter; Jones, Arwyn T

    2015-12-01

    A major unmet clinical need is a universal method for subcellular targeting of bioactive molecules to lysosomes. Delivery to this organelle enables either degradation of oncogenic receptors that are overexpressed in cancers, or release of prodrugs from antibody-drug conjugates. Here, we describe a general method that uses receptor crosslinking to trigger endocytosis and subsequently redirect trafficking of receptor:cargo complexes from their expected route, to lysosomes. By incubation of plasma membrane receptors with biotinylated cargo and subsequent addition of streptavidin to crosslink receptor:cargo-biotin complexes, we achieved rapid and selective lysosomal targeting of transferrin, an anti-MHC class I antibody, and the clinically approved anti-Her2 antibody trastuzumab. These three protein ligands each target a receptor with a distinct cellular function and intracellular trafficking profile. Importantly, we confirmed that crosslinking of trastuzumab increased lysosomal degradation of its cognate oncogenic receptor Her2 in breast cancer cell lines SKBR3 and BT474. These data suggest that crosslinking could be exploited for a wide range of target receptors, for navigating therapeutics through the endolysosomal pathway, for significant therapeutic benefit. PMID:26412588

  18. Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes

    PubMed Central

    Moody, Paul R; Sayers, Edward J; Magnusson, Johannes P; Alexander, Cameron; Borri, Paola; Watson, Peter; Jones, Arwyn T

    2015-01-01

    A major unmet clinical need is a universal method for subcellular targeting of bioactive molecules to lysosomes. Delivery to this organelle enables either degradation of oncogenic receptors that are overexpressed in cancers, or release of prodrugs from antibody–drug conjugates. Here, we describe a general method that uses receptor crosslinking to trigger endocytosis and subsequently redirect trafficking of receptor:cargo complexes from their expected route, to lysosomes. By incubation of plasma membrane receptors with biotinylated cargo and subsequent addition of streptavidin to crosslink receptor:cargo–biotin complexes, we achieved rapid and selective lysosomal targeting of transferrin, an anti-MHC class I antibody, and the clinically approved anti-Her2 antibody trastuzumab. These three protein ligands each target a receptor with a distinct cellular function and intracellular trafficking profile. Importantly, we confirmed that crosslinking of trastuzumab increased lysosomal degradation of its cognate oncogenic receptor Her2 in breast cancer cell lines SKBR3 and BT474. These data suggest that crosslinking could be exploited for a wide range of target receptors, for navigating therapeutics through the endolysosomal pathway, for significant therapeutic benefit. PMID:26412588

  19. An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease.

    PubMed

    Kizuka, Yasuhiko; Kitazume, Shinobu; Fujinawa, Reiko; Saito, Takashi; Iwata, Nobuhisa; Saido, Takaomi C; Nakano, Miyako; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro; Staufenbiel, Matthias; Hatsuta, Hiroyuki; Murayama, Shigeo; Manya, Hiroshi; Endo, Tamao; Taniguchi, Naoyuki

    2015-02-01

    The ?-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-? (A?) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of A?-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in A? plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics. PMID:25592972

  20. An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease

    PubMed Central

    Kizuka, Yasuhiko; Kitazume, Shinobu; Fujinawa, Reiko; Saito, Takashi; Iwata, Nobuhisa; Saido, Takaomi C; Nakano, Miyako; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro; Staufenbiel, Matthias; Hatsuta, Hiroyuki; Murayama, Shigeo; Manya, Hiroshi; Endo, Tamao; Taniguchi, Naoyuki

    2015-01-01

    The ?-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-? (A?) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of A?-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in A? plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics. PMID:25592972

  1. TNF? Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes.

    PubMed

    Hennigar, Stephen R; Kelleher, Shannon L

    2015-10-01

    Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-? (TNF?), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNF? retargets ZnT2 to lysosomes. We tested the hypothesis that TNF? signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNF? redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient?=?0.202??0.05 and 0.097??0.03; P<0.05) to lysosomes (0.292??0.03 and 0.649??0.03; P<0.0001), which increased lysosomal Zn (P<0.0001) and activated LCD (P<0.0001) compared to untreated cells. Mutation of the dileucine motif (L294V) eliminated the ability of TNF? to redistribute ZnT2 from late endosomes to lysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNF? increased (P<0.05) AP-3 binding to wt ZnT2 but not to L294V immunoprecipitates. Finally, using phospho- and dephospho-mimetics of predicted phosphorylation sites (T281, T288, and S296), we found that dephosphorylated S296 was required to target ZnT2 to accumulate Zn in lysosomes and activate LCD. Our findings suggest that women with variation in the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. PMID:25808614

  2. Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins

    PubMed Central

    Mark, Kevin G.; Simonetta, Marco; Maiolica, Alessio; Seller, Charles A.

    2014-01-01

    SUMMARY We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using Ligase Traps of eight different F-box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously uncharacterized substrates. Polyubiquitinated forms of candidate substrates associated with their cognate F-box partner, but not other Ligase Traps. Interestingly, the four most abundant candidate substrates identified for the F-box protein Saf1 were all vacuolar/lysosomal proteins. Analysis of one of these substrates, Prb1, showed that Saf1 selectively promotes ubiquitination of the unprocessed form of the zymogen. This suggests that Saf1 is part of a pathway that targets protein precursors for proteasomal degradation. PMID:24389104

  3. Plasticity of Polyubiquitin Recognition as Lysosomal Targeting Signals by the Endosomal Sorting Machinery

    PubMed Central

    Barriere, Herve; Nemes, Csilla; Du, Kai

    2007-01-01

    Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting. PMID:17686993

  4. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2? production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703712. PMID:19761405

  5. Lysosomal Targeting with Stable and Sensitive Fluorescent Probes (Superior LysoProbes): Applications for Lysosome Labeling and Tracking during Apoptosis

    PubMed Central

    Chen, Xin; Bi, Yue; Wang, Tianyang; Li, Pengfei; Yan, Xin; Hou, Shanshan; Bammert, Catherine E.; Ju, Jingfang; Gibson, K. Michael; Pavan, William J.; Bi, Lanrong

    2015-01-01

    Intracellular pH plays an important role in the response to cancer invasion. We have designed and synthesized a series of new fluorescent probes (Superior LysoProbes) with the capacity to label acidic organelles and monitor lysosomal pH. Unlike commercially available fluorescent dyes, Superior LysoProbes are lysosome-specific and are highly stable. The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy. Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis. Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes. PMID:25758662

  6. Impaired selection of invariant natural killer T cells in diverse mouse models of glycosphingolipid lysosomal storage diseases

    PubMed Central

    Gadola, Stephan D.; Silk, Jonathan D.; Jeans, Aruna; Illarionov, Petr A.; Salio, Mariolina; Besra, Gurdyal S.; Dwek, Raymond; Butters, Terry D.; Platt, Frances M.; Cerundolo, Vincenzo

    2006-01-01

    Glycolipid ligands for invariant natural killer T cells (iNKT cells) are loaded onto CD1d molecules in the late endosome/lysosome. Accumulation of glycosphingolipids (GSLs) in lysosomal storage diseases could potentially influence endogenous and exogenous lipid loading and/or presentation and, thus, affect iNKT cell selection or function. The percentages and frequency of iNKT cells were reduced in multiple mouse models of lysosomal GSL storage disease, irrespective of the specific genetic defect or lipid species stored. Reduced numbers of iNKT cells resulted in the absence of cytokine production in response to ?-galactosylceramide (?-GalCer) and reduced iNKT cellmediated lysis of wild-type targets loaded with ?-GalCer. The reduction in iNKT cells did not result from defective expression of CD1d or a lack of antigen-presenting cells. Although H-2 restricted CD4+ T cell responses were generally unaffected, processing of a lysosome-dependent analogue of ?-GalCer was impaired in all the strains of mice tested. These data suggest that GSL storage may result in alterations in thymic selection of iNKT cells caused by impaired presentation of selecting ligands. PMID:16982810

  7. Impaired selection of invariant natural killer T cells in diverse mouse models of glycosphingolipid lysosomal storage diseases.

    PubMed

    Gadola, Stephan D; Silk, Jonathan D; Jeans, Aruna; Illarionov, Petr A; Salio, Mariolina; Besra, Gurdyal S; Dwek, Raymond; Butters, Terry D; Platt, Frances M; Cerundolo, Vincenzo

    2006-10-01

    Glycolipid ligands for invariant natural killer T cells (iNKT cells) are loaded onto CD1d molecules in the late endosome/lysosome. Accumulation of glycosphingolipids (GSLs) in lysosomal storage diseases could potentially influence endogenous and exogenous lipid loading and/or presentation and, thus, affect iNKT cell selection or function. The percentages and frequency of iNKT cells were reduced in multiple mouse models of lysosomal GSL storage disease, irrespective of the specific genetic defect or lipid species stored. Reduced numbers of iNKT cells resulted in the absence of cytokine production in response to alpha-galactosylceramide (alpha-GalCer) and reduced iNKT cell-mediated lysis of wild-type targets loaded with alpha-GalCer. The reduction in iNKT cells did not result from defective expression of CD1d or a lack of antigen-presenting cells. Although H-2 restricted CD4(+) T cell responses were generally unaffected, processing of a lysosome-dependent analogue of alpha-GalCer was impaired in all the strains of mice tested. These data suggest that GSL storage may result in alterations in thymic selection of iNKT cells caused by impaired presentation of selecting ligands. PMID:16982810

  8. Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

    SciTech Connect

    Haylett, T.; Thilo, L.

    1986-10-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D/sub 1/, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from <0.1% to a steady-state level of approx.2.5% of the total label. As analyzed by NaDodSO/sub 4/ PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only approx.1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

  9. Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting.

    PubMed

    Joller, Nicole; Weber, Stefan S; Müller, Andreas J; Spörri, Roman; Selchow, Petra; Sander, Peter; Hilbi, Hubert; Oxenius, Annette

    2010-11-23

    The protective effect of antibodies (Abs) is generally attributed to neutralization or complement activation. Using Legionella pneumophila and Mycobacterium bovis bacillus Calmette-Guérin as a model, we discovered an additional mechanism of Ab-mediated protection effective against intracellular pathogens that normally evade lysosomal fusion. We show that Fc receptor (FcR) engagement by Abs, which can be temporally and spatially separated from bacterial infection, renders the host cell nonpermissive for bacterial replication and targets the pathogens to lysosomes. This process is strictly dependent on kinases involved in FcR signaling but not on host cell protein synthesis or protease activation. Based on these findings, we propose a mechanism whereby Abs and FcR engagement subverts the strategies by which intracellular bacterial pathogens evade lysosomal degradation. PMID:21048081

  10. Impairment of Lysosomal Activity as a Therapeutic Modality Targeting Cancer Stem Cells of Embryonal Rhabdomyosarcoma Cell Line RD

    PubMed Central

    Salerno, Manuela; Avnet, Sofia; Bonuccelli, Gloria; Hosogi, Shigekuni; Granchi, Donatella; Baldini, Nicola

    2014-01-01

    Rhabdomyosarcoma is the most frequent soft tissue sarcoma in children and adolescents, with a high rate of relapse that dramatically affects the clinical outcome. Multiagent chemotherapy, in combination with surgery and/or radiation therapy, is the treatment of choice. However, the relapse rate is disappointingly high and identification of new therapeutic tools is urgently needed. Under this respect, the selective block of key features of cancer stem cells (CSC) appears particularly promising. In this study, we isolated rhabdomyosarcoma CSC with stem-like features (high expression of NANOG and OCT3/4, self-renewal ability, multipotency). Rhabdomyosarcoma CSC showed higher invasive ability and a reduced cytotoxicity to doxorubicin in comparison to native cells, through a mechanism unrelated to the classical multidrug resistance process. This was dependent on a high level of lysosome acidity mediated by a high expression of vacuolar ATPase (V-ATPase). Since it was not associated with other paediatric cancers, like Ewings sarcoma and neuroblastoma, V-ATPase higher expression in CSC was rhabdomyosarcoma specific. Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole, or by specific siRNA silencing, significantly enhanced doxorubicin cytoxicity. Unexpectedly, lysosomal targeting also blocked cell growth and reduced the invasive potential of rhabdomyosarcoma CSC, even at very low doses of omeprazole (10 and 50 M, respectively). Based on these observations, we propose lysosome acidity as a valuable target to enhance chemosensitivity of rhabdomyosarcoma CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease. PMID:25329465

  11. Enhanced Human Epidermal Growth Factor Receptor 2 Degradation in Breast Cancer Cells by Lysosome-Targeting Gold Nanoconstructs.

    PubMed

    Lee, Hyojin; Dam, Duncan Hieu M; Ha, Ji Won; Yue, Jun; Odom, Teri W

    2015-10-27

    This paper describes how gold nanoparticle nanoconstructs can enhance anticancer effects of lysosomal targeting aptamers in breast cancer cells. Nanoconstructs consisting of anti-HER2 aptamer (human epidermal growth factor receptor 2, HApt) densely grafted on gold nanostars (AuNS) first targeted HER2 and then were internalized via HER2-mediated endocytosis. As incubation time increased, the nanoconstruct complexes were found in vesicular structures, starting from early endosomes to lysosomes as visualized by confocal fluorescence and differential interference contrast microscopy. Within the target organelle, lysosomes, HER2 was degraded by enzymes at low pH, which resulted in apoptosis. At specific time points related to the doubling time of the cancer cells, we found that accumulation of HER2-HApt-AuNS complexes in lysosomes, lysosomal activity, and lysosomal degradation of HER2 were positively correlated. Increased HER2 degradation by HApt-AuNS triggered cell death and cell cycle arrest in the G0/G1 phase inhibition of cell proliferation. This work shows how a perceived disadvantage of nanoparticle-based therapeutics-the inability of nanoconstructs to escape from vesicles and thus induce a biological response-can be overcome by both targeting lysosomes and exploiting lysosomal degradation of the biomarkers. PMID:26335372

  12. Novel lysosome targeted molecular transporter built on a guanidinium-poly-(propylene imine) hybrid dendron for efficient delivery of doxorubicin into cancer cells.

    PubMed

    Nair, Jyothi B; Mohapatra, Saswat; Ghosh, Surajit; Maiti, Kaustabh K

    2015-02-11

    An efficient synthetic approach has been adopted to construct a new dendron-based octa-guanidine appended molecular transporter with a lysosomal targeted peptide-doxorubicin conjugate. The transporter alone (G8-PPI-FL) is found to be non-toxic, showed higher cellular uptake compared to Arg-8-mer and exhibited excellent selectivity towards lysosomes in cathepsin B expressing HeLa cells, while the Dox-conjugate showed significant cytotoxicity to cancer cells without affecting the non-cancerous cells. PMID:25564099

  13. Novel lysosome targeted molecular transporter built on a guanidinium-poly-(propylene imine) hybrid dendron for efficient delivery of doxorubicin into cancer cells.

    TOXLINE Toxicology Bibliographic Information

    Nair JB; Mohapatra S; Ghosh S; Maiti KK

    2015-02-11

    An efficient synthetic approach has been adopted to construct a new dendron-based octa-guanidine appended molecular transporter with a lysosomal targeted peptide-doxorubicin conjugate. The transporter alone (G8-PPI-FL) is found to be non-toxic, showed higher cellular uptake compared to Arg-8-mer and exhibited excellent selectivity towards lysosomes in cathepsin B expressing HeLa cells, while the Dox-conjugate showed significant cytotoxicity to cancer cells without affecting the non-cancerous cells.

  14. Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis

    PubMed Central

    Napolitano, Gennaro; Johnson, Jennifer L; He, Jing; Rocca, Celine J; Monfregola, Jlenia; Pestonjamasp, Kersi; Cherqui, Stephanie; Catz, Sergio D

    2015-01-01

    Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS-deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns?/? mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns?/? mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload. PMID:25586965

  15. Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis.

    PubMed

    Napolitano, Gennaro; Johnson, Jennifer L; He, Jing; Rocca, Celine J; Monfregola, Jlenia; Pestonjamasp, Kersi; Cherqui, Stephanie; Catz, Sergio D

    2015-02-01

    Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS-deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns(-/-) mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns(-/-) mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload. PMID:25586965

  16. Selective labeling and monitoring pH changes of lysosomes in living cells with fluorogenic pH sensors.

    PubMed

    Ying, Lai-Qiang; Branchaud, Bruce P

    2011-06-15

    New BODIPY-based pH probes have been designed with excitation and emission wavelengths suitable for fluorescence microscopy and flow cytometry. These pH probes are cell-permeable, selectively label lysosomes, and can be used for noninvasive monitoring of lysosomal pH changes during physiological and pathological processes. PMID:21576021

  17. Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates.

    PubMed

    Sutherland, May S Kung; Sanderson, Russell J; Gordon, Kristine A; Andreyka, Jamie; Cerveny, Charles G; Yu, Changpu; Lewis, Timothy S; Meyer, Damon L; Zabinski, Roger F; Doronina, Svetlana O; Senter, Peter D; Law, Che-Leung; Wahl, Alan F

    2006-04-14

    The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics. PMID:16484228

  18. Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal Degradation

    PubMed Central

    Fielding, Ceri A.; Aicheler, Rebecca; Stanton, Richard J.; Wang, Eddie C. Y.; Han, Song; Seirafian, Sepehr; Davies, James; McSharry, Brian P.; Weekes, Michael P.; Antrobus, P. Robin; Prod'homme, Virginie; Blanchet, Fabien P.; Sugrue, Daniel; Cuff, Simone; Roberts, Dawn; Davison, Andrew J.; Lehner, Paul J.; Wilkinson, Gavin W. G.; Tomasec, Peter

    2014-01-01

    NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, ?? and ?? T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)16) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12US21; a genetic arrangement, which is suggestive of an accordion expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family. PMID:24787765

  19. Studies on the activity of selected lysosomal exoglycosidases in patients with systemic lupus erythematosus.

    PubMed

    Olczyk, K; Drózdz, M; Kucharz, E; Piwowarczyk, B; Kardaszewicz, E; Harbut-Gryłka, A

    1984-01-01

    The activity of selected lysosomal hydrolases ( (beta-galactosidase, beta-N-acetylglucosaminidase, beta-xylosidase, alpha-mannosidase, alpha-fucosidase) was determined in the blood serum of patients with systemic lupus erythematosus (SLE). An increased activity of all the enzymes studied was found as compared to control subjects. Presumable mechanisms of the observed changes are discussed. It is suggested that the studied enzymes may be responsible for the desintegration of the connective tissue ground substance in SLE. PMID:6436958

  20. Visualization of Endogenous and Exogenous Hydrogen Peroxide Using A Lysosome-Targetable Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Kim, Dabin; Kim, Gyoungmi; Nam, Sang-Jip; Yin, Jun; Yoon, Juyoung

    2015-02-01

    Reactive oxygen species (ROS) play crucial roles in diverse physiological processes; therefore, the efficient detection of ROS is very crucial. In this study, we report a boronate-based hydrogen peroxide (H2O2) probe having naphthalimide fluorophore. This probe also contained a morpholine moiety as a directing group for lysosome. The recognition property indicated that the probe exhibited high selectivity towards H2O2 not only in the solution but also in the living cells. Furthermore, it was used to monitor the level of endogenous and exogenous H2O2. These results support that the probe can function as an efficient indicator to detect H2O2.

  1. Burglar Target Selection

    PubMed Central

    Townsley, Michael; Bernasco, Wim; Ruiter, Stijn; Johnson, Shane D.; White, Gentry; Baum, Scott

    2015-01-01

    Objectives: This study builds on research undertaken by Bernasco and Nieuwbeerta and explores the generalizability of a theoretically derived offender target selection model in three cross-national study regions. Methods: Taking a discrete spatial choice approach, we estimate the impact of both environment- and offender-level factors on residential burglary placement in the Netherlands, the United Kingdom, and Australia. Combining cleared burglary data from all study regions in a single statistical model, we make statistical comparisons between environments. Results: In all three study regions, the likelihood an offender selects an area for burglary is positively influenced by proximity to their home, the proportion of easily accessible targets, and the total number of targets available. Furthermore, in two of the three study regions, juvenile offenders under the legal driving age are significantly more influenced by target proximity than adult offenders. Post hoc tests indicate the magnitudes of these impacts vary significantly between study regions. Conclusions: While burglary target selection strategies are consistent with opportunity-based explanations of offending, the impact of environmental context is significant. As such, the approach undertaken in combining observations from multiple study regions may aid criminology scholars in assessing the generalizability of observed findings across multiple environments. PMID:25866418

  2. Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation

    PubMed Central

    Wang, Wuyang; Gao, Qiong; Yang, Meimei; Zhang, Xiaoli; Yu, Lu; Lawas, Maria; Li, Xinran; Bryant-Genevier, Marthe; Southall, Noel T.; Marugan, Juan; Ferrer, Marc; Xu, Haoxing

    2015-01-01

    Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca2+ channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca2+ imaging and whole-lysosome patch clamping, lysosomal Ca2+ release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na+-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in NiemannPick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca2+ signaling. PMID:25733853

  3. Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation.

    PubMed

    Wang, Wuyang; Gao, Qiong; Yang, Meimei; Zhang, Xiaoli; Yu, Lu; Lawas, Maria; Li, Xinran; Bryant-Genevier, Marthe; Southall, Noel T; Marugan, Juan; Ferrer, Marc; Xu, Haoxing

    2015-03-17

    Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca(2+) channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca(2+) imaging and whole-lysosome patch clamping, lysosomal Ca(2+) release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na(+)-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann-Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca(2+) signaling. PMID:25733853

  4. Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture

    PubMed Central

    Kurt, Ramazan; Chandra, Partha K.; Aboulnasr, Fatma; Panigrahi, Rajesh; Ferraris, Pauline; Aydin, Yucel; Reiss, Krzysztof; Wu, Tong; Balart, Luis A.; Dash, Srikanta

    2015-01-01

    Background Hepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-?)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-? against HCV is impaired. Aim To investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment. Method HCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-?) and Type III IFN (IFN-?) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-? alone and IFN-? alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated. Results FFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-? antiviral response and HCV clearance. Type III IFN (IFN-?), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA). Conclusion Our study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1. PMID:25961570

  5. CDTI target selection criteria

    NASA Technical Reports Server (NTRS)

    Britt, C. L.; Davis, C. M.; Jackson, C. B.; Mcclellan, V. A.

    1984-01-01

    A Cockpit Display of Traffic Information (CDTI) is a cockpit instrument which provides information to the aircrew on the relative location of aircraft traffic in the vicinity of their aircraft (township). In addition, the CDTI may provide information to assist in navigation and in aircraft control. It is usually anticipated that the CDTI will be integrated with a horizontal situation indicator used for navigational purposes and/or with a weather radar display. In this study, several sets of aircraft traffic data are analyzed to determine statistics on the number of targets that will be displayed on a CDTI using various target selection criteria. Traffic data were obtained from an Atlanta Terminal Area Simulation and from radar tapes recorded at the Atlanta and Miami terminal areas. Results are given in the form of plots showing the average percentage of time (or probability) that an aircraft equipped with a CDTI would observe from 0 to 10 other aircraft on the display for range settings on the CDTI up to 30 n. mi. and using various target discrimination techniques.

  6. Rab7 Silencing Prevents ?-Opioid Receptor Lysosomal Targeting and Rescues Opioid Responsiveness to Strengthen Diabetic Neuropathic Pain Therapy

    PubMed Central

    Mousa, Shaaban A.; Shaqura, Mohammed; Khalefa, Baled I.; Zllner, Christian; Schaad, Laura; Schneider, Jonas; Shippenberg, Toni S.; Richter, Jan F.; Hellweg, Rainer; Shakibaei, Mehdi; Schfer, Michael

    2013-01-01

    Painful diabetic neuropathy is poorly controlled by analgesics and requires high doses of opioids, triggering side effects and reducing patient quality of life. This study investigated whether enhanced Rab7-mediated lysosomal targeting of peripheral sensory neuron ?-opioid receptors (MORs) is responsible for diminished opioid responsiveness in rats with streptozotocin-induced diabetes. In diabetic animals, significantly impaired peripheral opioid analgesia was associated with a loss in sensory neuron MOR and a reduction in functional MOR G-protein-coupling. In control animals, MORs were retained mainly on the neuronal cell membrane. In contrast, in diabetic rats, they were colocalized with upregulated Rab7 in LampI-positive perinuclear lysosome compartments. Silencing endogenous Rab7 with intrathecal Rab7-siRNA or, indirectly, by reversing nerve growth factor deprivation in peripheral sensory neurons not only prevented MOR targeting to lysosomes, restoring their plasma membrane density, but also rescued opioid responsiveness toward better pain relief. These findings elucidate in vivo the mechanisms by which enhanced Rab7 lysosomal targeting of MORs leads to a loss in opioid antinociception in diabetic neuropathic pain. This is in contrast to peripheral sensory neuron MOR upregulation and antinociception in inflammatory pain, and provides intriguing evidence that regulation of opioid responsiveness varies as a function of pain pathogenesis. PMID:23230081

  7. Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor.

    PubMed

    Zhao, Yuguang; Ren, Jingshan; Padilla-Parra, Sergi; Fry, Elizabeth E; Stuart, David I

    2014-01-01

    The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2-β-GCase lysosomal targeting. PMID:25027712

  8. Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor

    PubMed Central

    Zhao, Yuguang; Ren, Jingshan; Padilla-Parra, Sergi; Fry, Elizabeth E.; Stuart, David I.

    2014-01-01

    The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2–β-GCase lysosomal targeting. PMID:25027712

  9. Development of targetable two-photon fluorescent probes to image hypochlorous Acid in mitochondria and lysosome in live cell and inflamed mouse model.

    PubMed

    Yuan, Lin; Wang, Lu; Agrawalla, Bikram Keshari; Park, Sung-Jin; Zhu, Hai; Sivaraman, Balasubramaniam; Peng, Juanjuan; Xu, Qing-Hua; Chang, Young-Tae

    2015-05-13

    Hypochlorous acid (HOCl), as a highly potent oxidant, is well-known as a key "killer" for pathogens in the innate immune system. Recently, mounting evidence indicates that intracellular HOCl plays additional important roles in regulating inflammation and cellular apoptosis. However, the organelle(s) involved in the distribution of HOCl remain unknown, causing difficulty to fully exploit its biological functions in cellular signaling pathways and various diseases. One of the main reasons lies in the lack of effective chemical tools to directly detect HOCl at subcellular levels due to low concentration, strong oxidization, and short lifetime of HOCl. Herein, the first two-photon fluorescent HOCl probe (TP-HOCl 1) and its mitochondria- (MITO-TP) and lysosome- (LYSO-TP) targetable derivatives for imaging mitochondrial and lysosomal HOCl were reported. These probes exhibit fast response (within seconds), good selectivity, and high sensitivity (<20 nM) toward HOCl. In live cell experiments, both probes MITO-TP and LYSO-TP were successfully applied to detect intracellular HOCl in corresponding organelles. In particular, the two-photon imaging of MITO-TP and LYSO-TP in murine model shows that higher amount of HOCl can be detected in both lysosome and mitochondria of macrophage cells during inflammation condition. Thus, these probes could not only help clarify the distribution of subcellular HOCl, but also serve as excellent tools to exploit and elucidate functions of HOCl at subcellular levels. PMID:25905448

  10. AN UNCONVENTIONAL DILEUCINE AND A NOVEL CYTOSOLIC MOTIF ARE REQUIRED FOR THE LYSOSOMAL/MELANOSOMAL TARGETING OF OA1

    PubMed Central

    Piccirillo, Rosanna; Palmisano, Ilaria; Innamorati, Giulio; Bagnato, Paola; Altimare, Domenico; Schiaffino, Maria Vittoria

    2006-01-01

    SUMMARY The protein product of the gene responsible for ocular albinism type 1, named OA1, is a pigment cell-specific membrane glycoprotein, displaying features of G protein-coupled receptors, yet exclusively localized to late endosomes/lysosomes and melanosomes. To dissect the signals responsible for the intracellular localization of OA1 we generated LAMP1/OA1 chimeras and OA1 mutants at the cytosolic domains of the protein. By this approach we identified two separate sorting signals that are both necessary and sufficient for intracellular retention and lysosomal/melanosomal localization in melanocytic and non-melanocytic cells: an unconventional dileucine motif within the third cytosolic loop and a novel motif, characterized by a tryptophan-glutamic acid doublet, within the C-terminal tail. Both motifs must be mutated to promote the plasma membrane localization of OA1, suggesting that they can independently drive its intracellular targeting. In addition, both motifs act similarly as lysosomal sorting signals in non-melanocytic cells, but appear to carry different specificities in melanocytic cells. Our findings indicate that OA1 contains multiple unconventional signals responsible for its lysosomal/melanosomal localization, and reveal a remarkable and unforeseen complexity in the regulation of polytopic protein sorting to specialized secretory organelles. PMID:16621890

  11. Targeting (cellular) lysosomal acid ceramidase by B13: Design, synthesis and evaluation of novel DMG-B13 ester prodrugs

    PubMed Central

    Bai, Aiping; Szulc, Zdzislaw, M.; Bielawski, Jacek; Pierce, Jason S.; Rembisa, Barbara; Terzieva, Silva; Mao, Cungui; Xu, Ruijuan; Wu, Bill; Clarke, Christopher J.; Newcomb, Benjamin; Liu, Xiang; Norris, James; Hannun, Yusuf A.; Bielawska, Alicja

    2015-01-01

    Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N, N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13•HCl) and LCL596 (1-O-DMG-B13•HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13•2HCl) conjugates, were designed and synthesized through N, N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase. PMID:25456083

  12. Targeted rescue of a polycystic kidney disease mutation by lysosomal inhibition.

    PubMed

    Hofherr, Alexis; Wagner, Claudius J; Watnick, Terry; Köttgen, Michael

    2016-04-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal disease. The molecular pathogenesis of ADPKD is not completely known, and there is no approved therapy. To date, there is limited knowledge concerning the molecular consequences of specific disease-causing mutations. Here we show that the ADPKD missense variant TRPP2(D511V) greatly reduces TRPP2 protein stability, and that TRPP2(D511V) function can be rescued in vivo by small molecules targeting the TRPP2 degradation pathway. Expression of the TRPP2(D511V) protein was significantly reduced compared to wild-type TRPP2. Inhibition of lysosomal degradation of TRPP2(D511V) by the US Food and Drug Administration (FDA)-approved drug chloroquine strongly increased TRPP2 protein levels in vitro. The validation of these results in vivo requires appropriate animal models. However, there are currently no mouse models harboring human PKD2 missense mutations, and screening for chemical rescue of patient mutations in rodent models is time-consuming and expensive. Therefore, we developed a Drosophila melanogaster model expressing the ortholog of TRPP2(D511V) to test chemical rescue of mutant TRPP2 in vivo. Notably, chloroquine was sufficient to improve the phenotype of flies expressing mutant TRPP2. Thus, this proof-of-concept study highlights the potential of directed therapeutic approaches for ADPKD, and provides a rapid-throughput experimental model to screen PKD2 patient mutations and small molecules in vivo. PMID:26924047

  13. Naphthalimide-based fluorescent probe for selectively and specifically detecting glutathione in the lysosomes of living cells.

    PubMed

    Cao, Meijiao; Chen, Haiyan; Chen, Dan; Xu, Zhiqiang; Liu, Sheng Hua; Chen, Xiaoqiang; Yin, Jun

    2015-12-24

    A novel naphthalimide-based fluorescent probe employing a sulfonamide unit as a thiol-responsive group is reported. It is capable of efficiently distinguishing GSH from cysteine and homocysteine. Bioimaging shows that it has high selectivity in living cells and can visualize the level of GSH in lysosomes. It is worth mentioning that different groups on the imide unit can affect the selectivity and reaction dynamics of the probe towards thiols. PMID:26576682

  14. Targeting lysosomal degradation induces p53-dependent cell death and prevents cancer in mouse models of lymphomagenesis.

    PubMed

    Maclean, Kirsteen H; Dorsey, Frank C; Cleveland, John L; Kastan, Michael B

    2008-01-01

    Despite great interest in cancer chemoprevention, effective agents are few. Here we show that chloroquine, a drug that activates the stress-responsive Atm-p53 tumor-suppressor pathway, preferentially enhances the death of Myc oncogene-overexpressing primary mouse B cells and mouse embryonic fibroblasts (MEFs) and impairs Myc-induced lymphomagenesis in a transgenic mouse model of human Burkitt lymphoma. Chloroquine-induced cell death in primary MEFs and human colorectal cancer cells was dependent upon p53, but not upon the p53 modulators Atm or Arf. Accordingly, chloroquine impaired spontaneous lymphoma development in Atm-deficient mice, a mouse model of ataxia telangiectasia, but not in p53-deficient mice. Chloroquine treatment enhanced markers of both macroautophagy and apoptosis in MEFs but ultimately impaired lysosomal protein degradation. Interestingly, chloroquine-induced cell death was not dependent on caspase-mediated apoptosis, as neither overexpression of the antiapoptotic protein Bcl-2 nor deletion of the proapoptotic Bax and Bak affected chloroquine-induced MEF death. However, when both apoptotic and autophagic pathways were blocked simultaneously, chloroquine-induced killing of Myc-overexpressing cells was blunted. Thus chloroquine induces lysosomal stress and provokes a p53-dependent cell death that does not require caspase-mediated apoptosis. These findings specifically demonstrate that intermittent chloroquine use effectively prevents cancer in mouse models of 2 genetically distinct human cancer syndromes, Burkitt lymphoma and ataxia telangiectasia, suggesting that agents targeting lysosome-mediated degradation may be effective in cancer prevention. PMID:18097482

  15. Lysosomal Physiology

    PubMed Central

    Xu, Haoxing; Ren, Dejian

    2015-01-01

    Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases. PMID:25668017

  16. Biosynthesis, targeting, and processing of lysosomal proteins: pulse-chase labeling and immune precipitation.

    PubMed

    Pohl, Sandra; Hasilik, Andrej

    2015-01-01

    Incorporation of radioactive precursors of amino acids and/or modifier groups into proteins, isolation and sizing of polypeptide species of interest, and finally their detection and characterization provide a robust handle to examine the life cycle and varied modifications of any protein. A prerequisite in application of these techniques to lysosomal enzymes is the availability of an avid and specific antibody, because lysosomal proteins represent a very minor fraction of the cellular protein and must be purified without a significant loss many 1000-fold as conveniently as possible. Pulse-chase labeling and good knowledge on organelle-specific modifications of lysosomal proteins may enhance the information that can be obtained from such experiments. We describe procedures for pulse-chase labeling experiments that have proven to work with a commercially available antibody against a mouse and a human lysosomal protease and can be used as a reference in establishing the technique in any laboratory that has an access to a certified isotope facility and the knowledge to handle radioactivity safely. We discuss the crucial steps and refer to alternatives described in the literature. The present model protein cathepsin Z is synthesized as a larger proenzyme that contains two N-linked oligosaccharides and matures to a shorter single chain enzyme retaining the processed oligosaccharides. A pulse-chase experiment demonstrates the conversion of the precursor into the mature form. In addition, results on deglycosylation of metabolically labeled cathepsin Z are shown and the alterations in the apparent size of the glycopeptides are explained. PMID:25665441

  17. Interaction of HIV-1 Nef Protein with the Host Protein Alix Promotes Lysosomal Targeting of CD4 Receptor*

    PubMed Central

    Amorim, Nathaly A.; da Silva, Eullia M. L.; de Castro, Rodrigo O.; da Silva-Janurio, Mara E.; Mendona, Luiza M.; Bonifacino, Juan S.; da Costa, Luciana J.; daSilva, Luis L. P.

    2014-01-01

    Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. PMID:25118280

  18. Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor.

    PubMed

    Amorim, Nathaly A; da Silva, Eulália M L; de Castro, Rodrigo O; da Silva-Januário, Mara E; Mendonça, Luiza M; Bonifacino, Juan S; da Costa, Luciana J; daSilva, Luis L P

    2014-10-01

    Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. PMID:25118280

  19. Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone, 2-Acetylpyridine 4,4-Dimethyl-3-selenosemicarbazone (Ap44mSe): Lysosomal Membrane Permeabilization.

    PubMed

    Al-Eisawi, Zaynab; Stefani, Christian; Jansson, Patric J; Arvind, Akanksha; Sharpe, Philip C; Basha, Maram T; Iskander, George M; Kumar, Naresh; Kovacevic, Zaklina; Lane, Darius J R; Sahni, Sumit; Bernhardt, Paul V; Richardson, Des R; Kalinowski, Danuta S

    2016-01-14

    Selenosemicarbazones show marked antitumor activity. However, their mechanism of action remains unknown. We examined the medicinal chemistry of the selenosemicarbazone, 2-acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe), and its iron and copper complexes to elucidate its mechanisms of action. Ap44mSe demonstrated a pronounced improvement in selectivity toward neoplastic relative to normal cells compared to its parent thiosemicarbazone. It also effectively depleted cellular Fe, resulting in transferrin receptor-1 up-regulation, ferritin down-regulation, and increased expression of the potent metastasis suppressor, N-myc downstream regulated gene-1. Significantly, Ap44mSe limited deleterious methemoglobin formation, highlighting its usefulness in overcoming toxicities of clinically relevant thiosemicarbazones. Furthermore, Cu-Ap44mSe mediated intracellular reactive oxygen species generation, which was attenuated by the antioxidant, N-acetyl-l-cysteine, or Cu sequestration. Notably, Ap44mSe forms redox active Cu complexes that target the lysosome to induce lysosomal membrane permeabilization. This investigation highlights novel structure-activity relationships for future chemotherapeutic design and underlines the potential of Ap44mSe as a selective anticancer/antimetastatic agent. PMID:26645570

  20. Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders

    PubMed Central

    2014-01-01

    Background With over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling. Methods We have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls. Results We correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys. Conclusion We report the assessment of a nextgeneration-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis. PMID:24767253

  1. Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage.

    PubMed

    Du, H; Duanmu, M; Witte, D; Grabowski, G A

    1998-09-01

    Lysosomal acid lipase (LAL) is essential for the hydrolysis of the triglycerides and cholesteryl esters in lysosomes. Its deficiency produces two phenotypes, a severe infantile-onset variant, Wolman disease (WD), and a later onset variant, cholesteryl ester storage disease (CESD). A mouse model with a LAL null mutation was produced by targeting disruption of the mouse gene. Homozygote knockout mice (lal -/lal-) produce no LAL mRNA, protein or enzyme activity. The lal-/lal- mice are born in Mendelian ratios, are normal appearing at birth, and follow normal development into adulthood. However, massive accumulation of triglycerides and cholesteryl esters occurs in several organs. By 21 days, the liver develops a yellow-orange color and is approximately 1.5-2.0x larger than normal. The accumulated cholesteryl esters and triglycerides are approximately 30-fold greater than normal. The lal+/lal- mice have approximately 50% of normal LAL activity and do not show lipid accumulation. Male and female lal-/lal- mice are fertile and can be bred to produce progeny. This mouse model is a phenotypic model of human CESD, and a biochemical and histopathologic mimic of human WD. The lal-/lal- mice provide a model to determine the role of LAL in lipid metabolism and the pathogenesis of its deficiency states. PMID:9700186

  2. Biotherapeutic target or sink: analysis of the macrophage mannose receptor tissue distribution in murine models of lysosomal storage diseases.

    PubMed

    Zhang, Xin Sheen; Brondyk, William; Lydon, John T; Thurberg, Beth L; Piepenhagen, Peter A

    2011-06-01

    Lysosomal storage diseases (LSDs) are metabolic disorders caused by enzyme deficiencies that lead to lysosomal accumulation of undegraded substrates. Enzyme replacement therapies (ERT) have been developed as treatments for patients with Gaucher, Niemann-Pick, Fabry, and Pompe diseases. Depending on the disease, the corresponding therapeutic enzyme is designed to be internalized by diseased cells through receptor-mediated endocytosis via macrophage mannose receptors (MMR) or mannose-6-phosphate receptors (M6PR). Enzymes developed to treat Gaucher and Niemann-Pick diseases are meant to target MMR-expressing cells, and in the case of Cerezyme [recombinant human ?-glucocerebrosidase (rh?GC)] for treating Gaucher disease, glycans on the enzyme are modified to increase specificity toward this receptor. Due to heterogeneity in glycosylation on enzymes intended to target the M6PR, however, there may also be some unintended targeting to MMR-expressing cells, which could act as unwanted sinks. Examples include Fabrazyme [recombinant human ?-galactosidase A (rh?Gal)] for treating Fabry disease and Myozyme [recombinant human acid ?-glucosidase (rhGAA)] for treating Pompe disease. It is therefore of great interest to better understand the cell type and tissue distribution of MMR in murine LSD models used to evaluate ERT efficacy and mechanism of action. In this study, we generated affinity-purified polyclonal antibody against murine MMR and used it to carry out a systematic examination of MMR protein localization in murine models of Gaucher, Niemann-Pick, Fabry, and Pompe diseases. Using immunohistochemistry, immunofluorescence, and confocal microscopy, we examined MMR distribution in liver, spleen, lung, kidney, heart, diaphragm, quadriceps, and triceps in these animal models and compared them with MMR distribution in wild-type mice. PMID:21416197

  3. Lysosomal Targeting of E-Cadherin: a Unique Mechanism for the Down-Regulation of Cell-Cell Adhesion during Epithelial to Mesenchymal Transitions

    PubMed Central

    Palacios, Felipe; Tushir, Jogender S.; Fujita, Yasuyuki; D'Souza-Schorey, Crislyn

    2005-01-01

    A hallmark characteristic of epithelial tumor progression as well as some processes of normal development is the loss of the epithelial phenotype and acquisition of a motile or mesenchymal phenotype. Such epithelial to mesenchymal transitions are accompanied by the loss of E-cadherin function by either transcriptional or posttranscriptional mechanisms. Here we demonstrate that, upon v-Src expression, a potent trigger of epithelial to mesenchymal transitions, E-cadherin is internalized and then shuttled to the lysosome instead of being recycled back to the lateral membrane. Thus, while E-cadherin internalization facilitates the dissolution of adherens junctions, its subsequent traffic to the lysosome serves as a means to ensure that cells do not reform their cell-cell contacts and remain motile. We also show that ubiquitin tagging of E-cadherin is essential for its sorting to the lysosome. The lysosomal targeting of E-cadherin is mediated by hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and v-Src-induced activation of the Rab5 and Rab7 GTPases. Our studies reveal that the lysosomal targeting of E-cadherin is an important posttranscriptional mechanism to deplete cellular E-cadherin during Src-induced epithelial to mesenchymal transitions. PMID:15601859

  4. Lysosome electrophysiology.

    PubMed

    Zhong, Xi Z; Dong, Xian-Ping

    2015-01-01

    The physiology and functions of ion channels have been major topics of interest in biomedical research. Patch clamping is one of the most powerful techniques used in the study of ion channels and has been widely applied to the investigation of electrical properties of ion channels on the plasma membrane in a variety of cells. A number of ion channels have been found in intracellular lysosomal membranes. However, their properties had been difficult to study due to the lack of a direct patch-clamping methodology on lysosomal membranes. Past attempts to record lysosomal channels that were forced to express on the plasma membrane or reconstituted into lipid bilayers have largely generated inconclusive and conflicting results. Recently, a novel lysosome patch-clamping technique has been developed, making it possible to examine lysosomal channels under near physiological conditions. This chapter provides a detailed description of this technique, which has been successfully applied in several studies concerning lysosomal ion channels. This technique will expand our understanding of the nature of lysosomes and lysosome-related diseases. PMID:25665447

  5. A ruthenium(II) complex-based lysosome-targetable multisignal chemosensor for in vivo detection of hypochlorous acid.

    PubMed

    Cao, Liyan; Zhang, Run; Zhang, Wenzhu; Du, Zhongbo; Liu, Chunjun; Ye, Zhiqiang; Song, Bo; Yuan, Jingli

    2015-11-01

    Although considerable efforts have been made for the development of ruthenium(II) complex-based chemosensors and bioimaging reagents, the multisignal chemosensor using ruthenium(II) complexes as the reporter is scarce. In addition, the mechanisms of cellular uptake of ruthenium(II)-based chemosensors and their intracellular distribution are ill-defined. Herein, a new ruthenium(II) complex-based multisignal chemosensor, Ru-Fc, is reported for the highly sensitive and selective detection of lysosomal hypochlorous acid (HOCl). Ru-Fc is weakly luminescent because the MLCT (metal-to-ligand charge transfer) state is corrupted by the efficient PET (photoinduced electron transfer) process from Fc (ferrocene) moiety to Ru(II) center. The cleavage of Fc moiety by a HOCl-induced specific reaction leads to elimination of PET, which re-establishes the MLCT state of the Ru(II) complex, accompanied by remarkable photoluminescence (PL) and electrochemiluminescence (ECL) enhancements. The result of MTT assay showed that the proposed chemosensor, Ru-Fc, was low cytotoxicity. The applicability of Ru-Fc for the quantitative detection of HOCl in live cells was demonstrated by the confocal microscopy imaging and flow cytometry analysis. Dye colocalization studies confirmed very precise distribution of the Ru(II) complex in lysosomes, and inhibition studies revealed that the caveolae-mediated endocytosis played an important role during the cellular internalization of Ru-Fc. By using Ru-Fc as a chemosensor, the imaging of the endogenous HOCl generated in live macrophage cells during the stimulation was achieved. Furthermore, the practical applicability of Ru-Fc was demonstrated by the visualizing of HOCl in laboratory model animals, Daphnia magna and zebrafish. PMID:26256295

  6. The biogenesis of lysosomes and lysosome-related organelles.

    PubMed

    Luzio, J Paul; Hackmann, Yvonne; Dieckmann, Nele M G; Griffiths, Gillian M

    2014-09-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  7. Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection

    PubMed Central

    Lu, Feiran; Liang, Qiren; Abi-Mosleh, Lina; Das, Akash; De Brabander, Jef K; Goldstein, Joseph L; Brown, Michael S

    2015-01-01

    Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry. DOI: http://dx.doi.org/10.7554/eLife.12177.001 PMID:26646182

  8. The mechanical activation of mTOR signaling: an emerging role for late endosome/lysosomal targeting.

    PubMed

    Jacobs, Brittany L; Goodman, Craig A; Hornberger, Troy A

    2014-02-01

    It is well recognized that mechanical signals play a critical role in the regulation of skeletal muscle mass, and the maintenance of muscle mass is essential for mobility, disease prevention and quality of life. Furthermore, over the last 15 years it has become established that signaling through a protein kinase called the mammalian (or mechanistic) target of rapamycin (mTOR) is essential for mechanically-induced changes in protein synthesis and muscle mass, however, the mechanism(s) via which mechanical stimuli regulate mTOR signaling have not been defined. Nonetheless, advancements are being made, and an emerging body of evidence suggests that the late endosome/lysosomal (LEL) system might play a key role in this process. Therefore, the purpose of this review is to summarize this body of evidence. Specifically, we will first explain why the Ras homologue enriched in brain (Rheb) and phosphatidic acid (PA) are considered to be direct activators of mTOR signaling. We will then describe the process of endocytosis and its involvement in the formation of LEL structures, as well as the evidence which indicates that mTOR and its direct activators (Rheb and PA) are all enriched at the LEL. Finally, we will summarize the evidence that has implicated the LEL in the regulation of mTOR by various growth regulatory inputs such as amino acids, growth factors and mechanical stimuli. PMID:24162376

  9. Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.

    PubMed

    Lu, Feiran; Liang, Qiren; Abi-Mosleh, Lina; Das, Akash; De Brabander, Jef K; Goldstein, Joseph L; Brown, Michael S

    2015-01-01

    Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry. PMID:26646182

  10. Quasar target selection fiber efficiency

    SciTech Connect

    Newberg, H.; Yanny, B.

    1996-05-01

    We present estimates of the efficiency for finding QSOs as a function of limiting magnitude and galactic latitude. From these estimates, we have formulated a target selection strategy that should net 80,000 QSOs in the north galactic cap with an average of 70 fibers per plate, not including fibers reserved for high-redshift quasars. With this plan, we expect 54% of the targets to be QSOs. The North Galactic Cap is divided into two zones of high and low stellar density. We use about five times as many fibers for QSO candidates in the half of the survey with the lower stellar density as we use in the half with higher stellar density. The current plan assigns 15% of the fibers to FIRST radio sources; if these are not available, those fibers would be allocated to lower probability QSO sources, dropping the total number of QSOs by a small factor (5%). We will find about 17,000 additional quasars in the southern strips, and maybe a few more at very high redshift. Use was made of two data sets: the star and quasar simulated test data generated by Don Schneider, and the data from UJFN plate surveys by Koo (1986) and Kron (1980). This data was compared to results from the Palomar-Green Survey and a recent survey by Pat Osmer and collaborators.

  11. Expression and lysosomal targeting of CLN7, a major facilitator superfamily transporter associated with variant late-infantile neuronal ceroid lipofuscinosis

    PubMed Central

    Sharifi, A.; Kousi, M.; Sagn, C.; Bellenchi, G.C.; Morel, L.; Darmon, M.; H?lkov, H.; Ruivo, R.; Debacker, C.; El Mestikawy, S.; Elleder, M.; Lehesjoki, A.-E.; Jalanko, A.; Gasnier, B.; Kyttl, A.

    2010-01-01

    Neuronal ceroid lipofuscinoses (NCLs) constitute a group of progressive neurodegenerative disorders resulting from mutations in at least eight different genes. Mutations in the most recently identified NCL gene, MFSD8/CLN7, underlie a variant of late-infantile NCL (vLINCL). The MFSD8/CLN7 gene encodes a polytopic protein with unknown function, which shares homology with ion-coupled membrane transporters. In this study, we confirmed the lysosomal localization of the native CLN7 protein. This localization of CLN7 is not impaired by the presence of pathogenic missense mutations or after genetic ablation of the N-glycans. Expression of chimeric and full-length constructs showed that lysosomal targeting of CLN7 is mainly determined by an N-terminal dileucine motif, which specifically binds to the heterotetrameric adaptor AP-1 in vitro. We also show that CLN7 mRNA is more abundant in neurons than astrocytes and microglia, and that it is expressed throughout rat brain, with increased levels in the granular layer of cerebellum and hippocampal pyramidal cells. Interestingly, this cellular and regional distribution is in good agreement with the autofluorescent lysosomal storage and cell loss patterns found in brains from CLN7-defective patients. Overall, these data highlight lysosomes as the primary site of action for CLN7, and suggest that the pathophysiology underpinning CLN7-associated vLINCL is a cell-autonomous process. PMID:20826447

  12. Expression and lysosomal targeting of CLN7, a major facilitator superfamily transporter associated with variant late-infantile neuronal ceroid lipofuscinosis.

    PubMed

    Sharifi, A; Kousi, M; Sagné, C; Bellenchi, G C; Morel, L; Darmon, M; Hulková, H; Ruivo, R; Debacker, C; El Mestikawy, S; Elleder, M; Lehesjoki, A-E; Jalanko, A; Gasnier, B; Kyttälä, A

    2010-11-15

    Neuronal ceroid lipofuscinoses (NCLs) constitute a group of progressive neurodegenerative disorders resulting from mutations in at least eight different genes. Mutations in the most recently identified NCL gene, MFSD8/CLN7, underlie a variant of late-infantile NCL (vLINCL). The MFSD8/CLN7 gene encodes a polytopic protein with unknown function, which shares homology with ion-coupled membrane transporters. In this study, we confirmed the lysosomal localization of the native CLN7 protein. This localization of CLN7 is not impaired by the presence of pathogenic missense mutations or after genetic ablation of the N-glycans. Expression of chimeric and full-length constructs showed that lysosomal targeting of CLN7 is mainly determined by an N-terminal dileucine motif, which specifically binds to the heterotetrameric adaptor AP-1 in vitro. We also show that CLN7 mRNA is more abundant in neurons than astrocytes and microglia, and that it is expressed throughout rat brain, with increased levels in the granular layer of cerebellum and hippocampal pyramidal cells. Interestingly, this cellular and regional distribution is in good agreement with the autofluorescent lysosomal storage and cell loss patterns found in brains from CLN7-defective patients. Overall, these data highlight lysosomes as the primary site of action for CLN7, and suggest that the pathophysiology underpinning CLN7-associated vLINCL is a cell-autonomous process. PMID:20826447

  13. TFEB regulates lysosomal proteostasis.

    PubMed

    Song, Wensi; Wang, Fan; Savini, Marzia; Ake, Ashley; di Ronza, Alberto; Sardiello, Marco; Segatori, Laura

    2013-05-15

    Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a β-hexosaminidase mutant associated with the development of another LSD, Tay-Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs. PMID:23393155

  14. DRUG INDUCED PHOSPHOLIPIDOSIS: AN ACQUIRED LYSOSOMAL STORAGE DISORDER

    PubMed Central

    Shayman, James A.; Abe, Akira

    2012-01-01

    There is a strong association between lysosome enzyme deficiencies and monogenic disorders resulting in lysosomal storage disease. Of the more than 75 characterized lysosomal proteins, two thirds are directly linked to inherited diseases of metabolism. Only one lysosomal storage disease, Niemann-Pick disease, is associated with impaired phospholipid metabolism. However, other phospholipases are found in the lysosome but remain poorly characterized. A recent exception is lysosomal phospholipase A2 (group XV phospholipase A2). Although no inherited disorder of lysosomal phospholipid metabolism has yet been associated with a loss of function of this lipase, this enzyme may be a target for an acquired form of lysosomal storage, drug induced phospholipidosis. PMID:22960355

  15. A cytotoxic type III secretion effector of Vibrio parahaemolyticus targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes.

    PubMed

    Matsuda, Shigeaki; Okada, Natsumi; Kodama, Toshio; Honda, Takeshi; Iida, Tetsuya

    2012-01-01

    Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H(+)-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepA?C), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death. PMID:22829766

  16. Lysosomal Trafficking Regulator (LYST).

    PubMed

    Ji, Xiaojie; Chang, Bo; Naggert, Jrgen K; Nishina, Patsy M

    2016-01-01

    Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections. PMID:26427484

  17. Selective Targeting to Glioma with Nucleic Acid Aptamers

    PubMed Central

    Aptekar, Shraddha; Arora, Mohit; Lawrence, Clare Louise; Lea, Robert William; Ashton, Katherine; Dawson, Tim; Alder, Jane Elizabeth; Shaw, Lisa

    2015-01-01

    Malignant glioma is characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue; hence, diagnosis and treatment is difficult and patient survival is poor. Aptamers contribute a promising and unique technology for the in vitro imaging of live cells and tissues, with a potentially bright future in clinical diagnostics and therapeutics for malignant glioma. The binding selectivity, uptake capacity and binding target of two DNA aptamers, SA43 and SA44, were investigated in glioma cells and patient tissues. The binding assay showed that SA43 and SA44 bound with strong affinity (Kd, 21.56 ± 4.60 nM and Kd, 21.11 ± 3.30 nM respectively) to the target U87MG cells. Quantitative analysis by flow cytometry showed that the aptamers were able to actively internalise in U87MG and 1321N1 glioma cells compared to the non-cancerous and non-glioma cell types. Confocal microscopy confirmed staining in the cytoplasm, and co-localisation studies with endoplasmic reticulum, Golgi apparatus and lysosomal markers suggested internalisation and compartmentalisation within the endomembrane system. Both aptamers selectively bound to Ku 70 and Ku 80 DNA repair proteins as determined by aptoprecipitation (AP) followed by mass spectrometry analysis and confirmation by Western blot. In addition, aptohistochemical (AHC) staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was significantly higher for SA43 aptamer in glioma tissues (grade I, II, III and IV) compared to the non-cancerous tissues, whereas SA44 did not show selectivity towards glioma tissues. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, shows promise for histological diagnosis of glioma. PMID:26252900

  18. Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis

    PubMed Central

    Nawaratna, Sujeevi S. K.; Gobert, Geoffrey N.; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P.; Jones, Malcolm K.

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  19. Lysosome-associated membrane glycoprotein (LAMP)--preliminary study on a hidden antigen target for vaccination against schistosomiasis.

    PubMed

    Nawaratna, Sujeevi S K; Gobert, Geoffrey N; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P; Jones, Malcolm K

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16-25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  20. Target Selection for the TESS Mission

    NASA Astrophysics Data System (ADS)

    Pepper, Joshua; Stassun, Keivan

    2015-12-01

    The goal of the TESS mission is to discover small, rocky planets transiting bright stars. To reach that goal, we have constructed a compiled catalog of stars from which to select TESS targets. The catalog contains all dwarf stars in the sky with spectral types F5 and later, and I < 12, along with selected sets of fainter M stars. Provisions are being made to augment the target list with stars that fall outside the nominal spectral type and magnitude limits, and to permit dynamic updating of the catalog to accommodate new survey data being released (e.g. Gaia). I will describe the overall target selection strategy, the current target catalog, and how we intend to further expand and refine the TESS target lists.

  1. SILAC-based quantitative proteomics identified lysosome as a fast response target to PDT agent Gd-N induced oxidative stress in human ovarian cancer IGROV1 cells.

    PubMed

    Qi, Dandan; Wang, Qianqian; Li, Hongguang; Zhang, Tao; Lan, Rongfeng; Kwong, Daniel W J; Wong, Wai-Kwok; Wong, Ka-Leung; Li, Shuiming; Lu, Fei

    2015-11-01

    Biological systems have developed an intact network and strategies in response to various environmental pressures such as irradiation, viral invasion and oxidative stress. Therefore, elucidation of the cellular response mechanism toward oxidative stress can contribute to the knowledge of redox regulation. By using a newly developed gadolinium based photodynamic therapy (PDT) agent Gd-N and SILAC quantified proteomic analysis, we observed 485 proteins dysregulated in expression, 106 in phosphorylation and 1050 in oxidation. Interestingly, lysosome was discovered as the main organelle affected by Gd-N induced singlet oxygen, along with the down regulation of a majority of lysosomal acid hydrolases and proton pump complex ATP6V/TCIRG1. Besides, phosphorylation sites with sequence patterns "TP" or "SP" were enriched in dysregulated phosphoproteins. Protein oxidation also shows sequence patterns in target proteins with "M.D" or "KM" taking methionine as the central residue. Oxidized proteins were most enriched in the pathways of Parkinson's disease, an oxidative stress closely related neurodegenerative disease. In conclusion, our study reveals new insights into the cellular mechanism to oxidative stress and may contribute to the discovery of new targets and development of novel PDT agents. PMID:26331702

  2. Lysosomal dysfunction causes neurodegeneration in mucolipidosis II knock-in mice

    PubMed Central

    Kollmann, K.; Damme, M.; Markmann, S.; Morelle, W.; Schweizer, M.; Hermans-Borgmeyer, I.; Rchert, A. K.; Pohl, S.; Lbke, T.; Michalski, J.-C.; Kkel, R.; Walkley, S. U.

    2012-01-01

    Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated knock-in mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as ?-l-fucosidase, ?-hexosaminidase, ?-mannosidase or NiemannPick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease. PMID:22961545

  3. Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages

    NASA Technical Reports Server (NTRS)

    Lah, T. T.; Hawley, M.; Rock, K. L.; Goldberg, A. L.

    1995-01-01

    Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

  4. [Application of lysosomal detection in marine pollution monitoring: research progress].

    PubMed

    Weng, You-Zhu; Fang, Yong-Qiang; Zhang, Yu-Sheng

    2013-11-01

    Lysosome is an important organelle existing in eukaryotic cells. With the development of the study on the structure and function of lysosome in recent years, lysosome is considered as a target of toxic substances on subcellular level, and has been widely applied abroad in marine pollution monitoring. This paper summarized the biological characteristics of lysosomal marker enzyme, lysosome-autophagy system, and lysosomal membrane, and introduced the principles and methods of applying lysosomal detection in marine pollution monitoring. Bivalve shellfish digestive gland and fish liver are the most sensitive organs for lysosomal detection. By adopting the lysosomal detection techniques such as lysosomal membrane stability (LMS) test, neutral red retention time (NRRT) assay, morphological measurement (MM) of lysosome, immunohistochemical (Ih) assay of lysosomal marker enzyme, and electron microscopy (EM), the status of marine pollution can be evaluated. It was suggested that the lysosome could be used as a biomarker for monitoring marine environmental pollution. The advantages and disadvantages of lysosomal detection and some problems worthy of attention were analyzed, and the application prospects of lysosomal detection were discussed. PMID:24564165

  5. Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

    PubMed Central

    Maejima, Ikuko; Takahashi, Atsushi; Omori, Hiroko; Kimura, Tomonori; Takabatake, Yoshitsugu; Saitoh, Tatsuya; Yamamoto, Akitsugu; Hamasaki, Maho; Noda, Takeshi; Isaka, Yoshitaka; Yoshimori, Tamotsu

    2013-01-01

    Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis. PMID:23921551

  6. Regulated lysosomal exocytosis mediates cancer progression

    PubMed Central

    Machado, Eda; White-Gilbertson, Shai; van de Vlekkert, Diantha; Janke, Laura; Moshiach, Simon; Campos, Yvan; Finkelstein, David; Gomero, Elida; Mosca, Rosario; Qiu, Xiaohui; Morton, Christopher L.; Annunziata, Ida; d’Azzo, Alessandra

    2015-01-01

    Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf−⁄− mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance. PMID:26824057

  7. PIPing on lysosome tubes

    PubMed Central

    Ktistakis, Nicholas T; Tooze, Sharon A

    2013-01-01

    EMBO J (2013) 32, 324339 doi:10.1038/emboj.2012.341; published online 12212012 The role of lysosomes in important cellular responses, including phagocytosis, cell surface repair, and autophagy underlies a number of human diseases. Furthermore, the role of the lysosomal surface in TORC1 signalling has revealed unexpected properties of these organelles. In this issue, Sridhar et al (2013) uncover an important role for PI(4)P for lysosome function under normal nutrient conditions and after prolonged nutrient deprivation. Ana Maria Cuervo, the late Dennis Shields, and colleagues (Sridhar et al, 2013) conclude that PI4 kinase III? on the surface of the lysosome controls the fidelity of sorting from the lysosome, and is required for autophagic lysosome reformation (ALR). These novel findings provide important insights into the complexities of the lipid composition of the lysosome, and how these lipids may control lysosome function. PMID:23314746

  8. Structural Implications for Selective Targeting of PARPs

    PubMed Central

    Steffen, Jamin D.; Brody, Jonathan R.; Armen, Roger S.; Pascal, John M.

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD+ as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed synthetic lethality. In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients. PMID:24392349

  9. Lysosomes as mediators of drug resistance in cancer.

    PubMed

    Zhitomirsky, Benny; Assaraf, Yehuda G

    2016-01-01

    Drug resistance remains a leading cause of chemotherapeutic treatment failure and cancer-related mortality. While some mechanisms of anticancer drug resistance have been well characterized, multiple mechanisms remain elusive. In this respect, passive ion trapping-based lysosomal sequestration of multiple hydrophobic weak-base chemotherapeutic agents was found to reduce the accessibility of these drugs to their target sites, resulting in a markedly reduced cytotoxic effect and drug resistance. Recently we have demonstrated that lysosomal sequestration of hydrophobic weak base drugs triggers TFEB-mediated lysosomal biogenesis resulting in an enlarged lysosomal compartment, capable of enhanced drug sequestration. This study further showed that cancer cells with an increased number of drug-accumulating lysosomes are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. In addition to passive drug sequestration of hydrophobic weak base chemotherapeutics, other mechanisms of lysosome-mediated drug resistance have also been reported; these include active lysosomal drug sequestration mediated by ATP-driven transporters from the ABC superfamily, and a role for lysosomal copper transporters in cancer resistance to platinum-based chemotherapeutics. Furthermore, lysosomal exocytosis was suggested as a mechanism to facilitate the clearance of chemotherapeutics which highly accumulated in lysosomes, thus providing an additional line of resistance, supplementing the organelle entrapment of chemotherapeutics away from their target sites. Along with these mechanisms of lysosome-mediated drug resistance, several approaches were recently developed for the overcoming of drug resistance or exploiting lysosomal drug sequestration, including lysosomal photodestruction and drug-induced lysosomal membrane permeabilization. In this review we explore the current literature addressing the role of lysosomes in mediating cancer drug resistance as well as novel modalities to overcome this chemoresistance. PMID:26830313

  10. Target selection for the HRIBF Project

    SciTech Connect

    Dellwo, J.; Alton, G.D.; Batchelder, J.C.

    1994-12-31

    Experiments are in progress at the Oak Ridge National Laboratory (ORNL) which are designed to select the most appropriate target materials for generating particular radioactive ion beams for the Holifield Radioactive Ion Beam Facility (HRIBF). The 25-MV tandem accelerator is used to implant stable complements of interesting radioactive elements into refractory targets mounted in a high-temperature FEBIAD ion source which is on-line at the UNISOR facility. These experiments permit selection of the target material most appropriate for the rapid release of the element of interest, as well as realistic estimates of the efficiency of the FEBIAD source. From diffusion release data information on the release times and diffusion coefficients can be derived. Diffusion coefficients for CI implanted into and diffused from CeS and Zr{sub 5}Si{sub 3} and As, Br, and Se implanted into and diffused from Zr{sub 5}Ge{sub 3} have been derived from the resulting intensity versus time profiles.

  11. Spectral Signatures of Saccade Target Selection.

    PubMed

    Carl, Christine; Hipp, Joerg F; König, Peter; Engel, Andreas K

    2016-01-01

    Action generation relies on a widely distributed network of brain areas. However, little is known about the spatiotemporal dynamics of neuronal activity in the network that gives rise to voluntary action in humans. Here, we used magnetoencephalography (MEG) and source analysis (n = 15, 7 female subjects) to investigate the spectral signatures of human cortical networks engaged in active and intrinsically motivated viewing behavior. We compared neuronal activity of externally cued saccades with saccades to freely chosen targets. For planning and execution of both saccade types, we found an increase in gamma band (~64-128 Hz) activity and a concurrent decrease in beta band (~12-32 Hz) activity in saccadic control areas, including the intraparietal sulcus and the frontal eye fields. Guided compared to voluntary actions were accompanied by stronger transient increases in the gamma and low frequency (<16 Hz) range immediately following the instructional cue. In contrast, action selection between competing alternatives was reflected by stronger sustained fronto-parietal gamma increases that occurred later in time and persisted until movement execution. This sustained enhancement for free target selection was accompanied by a spatially widespread reduction of lower frequency power (~8-45 Hz) in parietal and extrastriate areas. Our results suggest that neuronal population activity in the gamma frequency band in a distributed network of fronto-parietal areas reflects the intrinsically driven process of selection among competing behavioral alternatives. PMID:25690830

  12. MaNGA: Target selection and Optimization

    NASA Astrophysics Data System (ADS)

    Wake, David

    2015-01-01

    The 6-year SDSS-IV MaNGA survey will measure spatially resolved spectroscopy for 10,000 nearby galaxies using the Sloan 2.5m telescope and the BOSS spectrographs with a new fiber arrangement consisting of 17 individually deployable IFUs. We present the simultaneous design of the target selection and IFU size distribution to optimally meet our targeting requirements. The requirements for the main samples were to use simple cuts in redshift and magnitude to produce an approximately flat number density of targets as a function of stellar mass, ranging from 1x109 to 1x1011 M⊙, and radial coverage to either 1.5 (Primary sample) or 2.5 (Secondary sample) effective radii, while maximizing S/N and spatial resolution. In addition we constructed a 'Color-Enhanced' sample where we required 25% of the targets to have an approximately flat number density in the color and mass plane. We show how these requirements are met using simple absolute magnitude (and color) dependent redshift cuts applied to an extended version of the NASA Sloan Atlas (NSA), how this determines the distribution of IFU sizes and the resulting properties of the MaNGA sample.

  13. MaNGA: Target selection and Optimization

    NASA Astrophysics Data System (ADS)

    Wake, David

    2016-01-01

    The 6-year SDSS-IV MaNGA survey will measure spatially resolved spectroscopy for 10,000 nearby galaxies using the Sloan 2.5m telescope and the BOSS spectrographs with a new fiber arrangement consisting of 17 individually deployable IFUs. We present the simultaneous design of the target selection and IFU size distribution to optimally meet our targeting requirements. The requirements for the main samples were to use simple cuts in redshift and magnitude to produce an approximately flat number density of targets as a function of stellar mass, ranging from 1x109 to 1x1011 M⊙, and radial coverage to either 1.5 (Primary sample) or 2.5 (Secondary sample) effective radii, while maximizing S/N and spatial resolution. In addition we constructed a "Color-Enhanced" sample where we required 25% of the targets to have an approximately flat number density in the color and mass plane. We show how these requirements are met using simple absolute magnitude (and color) dependent redshift cuts applied to an extended version of the NASA Sloan Atlas (NSA), how this determines the distribution of IFU sizes and the resulting properties of the MaNGA sample.

  14. Parkin-mediated K63-polyubiquitination targets ubiquitin C-terminal hydrolase L1 for degradation by the autophagy-lysosome system.

    PubMed

    McKeon, Jeanne E; Sha, Di; Li, Lian; Chin, Lih-Shen

    2015-05-01

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a key neuronal deubiquitinating enzyme which is mutated in Parkinson disease (PD) and in childhood-onset neurodegenerative disorder with optic atrophy. Furthermore, reduced UCH-L1 protein levels are associated with a number of neurodegenerative diseases, whereas up-regulation of UCH-L1 protein expression is found in multiple types of cancer. However, very little is known about how UCH-L1 protein level is regulated in cells. Here, we report that UCH-L1 is a novel interactor and substrate of PD-linked E3 ubiquitin-protein ligase parkin. We find that parkin mediates K63-linked polyubiquitination of UCH-L1 in cooperation with the Ubc13/Uev1a E2 ubiquitin-conjugating enzyme complex and promotes UCH-L1 degradation by the autophagy-lysosome pathway. Targeted disruption of parkin gene expression in mice causes a significant decrease in UCH-L1 ubiquitination with a concomitant increase in UCH-L1 protein level in brain, supporting an in vivo role of parkin in regulating UCH-L1 ubiquitination and degradation. Our findings reveal a direct link between parkin-mediated ubiquitin signaling and UCH-L1 regulation, and they have important implications for understanding the roles of these two proteins in health and disease. PMID:25403879

  15. 78 kDa receptor for Man6P-independent lysosomal enzyme targeting: Biosynthetic transport from endoplasmic reticulum to 'high-density vesicles'

    SciTech Connect

    Gonzalez-Noriega, Alfonso . E-mail: gonor@biomedicas.unam.mx; Ortega Cuellar, Daniel D.; Michalak, Colette

    2006-04-15

    Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH{sub 4}Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor.

  16. Optogenetic acidification of synaptic vesicles and lysosomes.

    PubMed

    Rost, Benjamin R; Schneider, Franziska; Grauel, M Katharina; Wozny, Christian; Bentz, Claudia G; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

    2015-12-01

    Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes. PMID:26551543

  17. Lysosomes, cholesterol and atherosclerosis

    PubMed Central

    Jerome, W Gray

    2011-01-01

    Cholesterol-engorged macrophage foam cells are a critical component of the atherosclerotic lesion. Reducing the sterol deposits in lesions reduces clinical events. Sterol accumulations within lysosomes have proven to be particularly hard to mobilize out of foam cells. Moreover, excess sterol accumulation in lysosomes has untoward effects, including a complete disruption of lysosome function. Recently, we demonstrated that treatment of sterol-engorged macrophages in culture with triglyceride-containing particles can reverse many of the effects of cholesterol on lysosomes and dramatically reduce the sterol burden in these cells. This article describes what is known about lysosomal sterol engorgement, discusses the possible mechanisms by which triglyceride could produce its effects, and evaluates the possible positive and negative effects of reducing the lysosomal cholesterol levels in foam cells. PMID:21643524

  18. Rabies DNA vaccine encoding lysosome-targeted glycoprotein supplemented with Emulsigen-D confers complete protection in preexposure and postexposure studies in BALB/c mice.

    PubMed

    Kaur, Manpreet; Saxena, Ankur; Rai, Anant; Bhatnagar, Rakesh

    2010-01-01

    The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for an improved rabies vaccine. Thus, DNA vaccine based on lysosome-targeted glycoprotein of the rabies virus was evaluated in BALB/c mice. It imparted partial protection (60%) against challenge with 20 LD(50) of the challenge virus standard (CVS) strain of rabies virus. To improve the outcome of vaccination, to ultimately enhance the immune response, we investigated different routes for DNA vaccine delivery, varied doses of DNA, and the influence of adjuvant supplementation. The highest immune response pertaining to IgG antibody titer, with a predominantly IgG1/IgG2a subclass distribution, effective cellular immunity, and a high level of rabies virus neutralizing antibodies (RVNAs) was attained by the optimized DNA vaccine formulation comprising intramuscular administration of 100 microg of DNA vaccine supplemented with Emulsigen-D. In preexposure prophylaxis, a 3-dose regimen of this formulation generated a high RVNA titer (32 IU/ml) and conferred complete protection against challenge with 20 LD(50) of CVS. For postexposure efficacy analysis, rabies was experimentally induced with 50 LD(50) of CVS. Subsequent therapy with 5 doses of the formulation completely prevented rabies in BALB/c mice, which maintained protective RVNA titers of 4 IU/ml. The World Health Organization recommended rabies protective titer threshold is 0.5 IU/ml. Thus, this optimized DNA vaccine formulation provides an avenue for preventing and controlling rabies. PMID:19741168

  19. Iron-dependent lysosomal dysfunction mediated by a natural product hybrid.

    PubMed

    Mariani, A; Mai, T T; Zacharioudakis, E; Hienzsch, A; Bartoli, A; Caeque, T; Rodriguez, R

    2016-01-14

    Artesumycin is a fluorescent hybrid of the natural products marmycin A and artemisinin. It was designed to combine the lysosomotropic properties of the angucycline and the iron-reactive capacity of the endoperoxide to target the lysosomal compartment of cancer cells. Herein, we show that artesumycin inhibits cancer cell proliferation in an iron-dependent manner and chemically fragments in vitro in the presence of redox-active iron(ii). Visual detection of artesumycin by fluorescence microscopy provided substantial evidence that the small molecule selectively targets lysosomes. This original approach based on a fluorescent and iron-reactive probe represents a powerful strategy for initiating and, concomitantly, visualizing lysosomal dysfunction in human cells. PMID:26645586

  20. Selective prothymocyte targeting by prenatal diethylstilbesterol exposure.

    PubMed

    Holladay, S D; Blaylock, B L; Comment, C E; Heindel, J J; Fox, W M; Korach, K S; Luster, M I

    1993-11-01

    Estrogens have been reported to modulate immunologic responses at both physiologic and pharmacologic concentrations. Treatment of experimental animals with the synthetic estrogen, diethylstilbesterol (DES), markedly decreases thymic cellularity, manifested histologically as a progressive loss of cortical thymic lymphocytes. In the present report thymic atrophy after prenatal DES exposure was found to be more severe than has been reported following adult exposure, indicating a possible greater sensitivity of the developing immune system to estrogenic hormones. DES exposure resulted in a limited alteration of cell maturation within the fetal thymus as evidenced by only slight alterations in the expression of CD4 and CD8 cell-surface antigens. To examine the possibility that DES targets hematopoietic stem cells in the fetal liver, cytometric analysis was conducted using a panel of fluorescent antibodies to quantitate the hematopoietic subpopulations present in control and DES-exposed Gestational Day (gd) 18 fetal mouse liver. There were no significant DES-induced alterations in the number of hematopoietic stem cells, or in fetal liver cells expressing CD44 (hematopoietic precursors), Mac-1 (granulocyte-macrophage lineage precursors), or CD45R (B-lineage lymphocytes) surface antigens. However, DES selectively reduced the number of fetal liver precursors containing the lymphocyte stem cell-specific DNA polymerase, terminal deoxynucleotidyl transferase, which suggested that DES may specifically target the fetal liver prothymocyte. Reconstitution of irradiated hosts with gd 18 fetal liver from vehicle and DES-exposed syngeneic donors demonstrated an impaired ability of the DES-treated fetal liver to repopulate the thymus of irradiated hosts. In addition, fetal liver cells enriched for prelymphoid cells contained potentially significant levels of estrogen specific receptors. Taken together these data, in conjunction with the lack of direct thymocyte injury (necrosis, apoptosis, and/or inhibition of cell proliferation) by DES treatment, suggest that estrogen-mediated thymic atrophy may result, at least in part, from a specific alteration in the lymphocyte stem cell population responsible for colonizing the thymus. PMID:8242756

  1. Lysosome Transport as a Function of Lysosome Diameter

    PubMed Central

    Bandyopadhyay, Debjyoti; Cyphersmith, Austin; Zapata, Jairo A.; Kim, Y. Joseph; Payne, Christine K.

    2014-01-01

    Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter. PMID:24497985

  2. Selecting a Targeting Method to Identify BPL Households in India

    ERIC Educational Resources Information Center

    Alkire, Sabina; Seth, Suman

    2013-01-01

    This paper proposes how to select a methodology to target multidimensionally poor households, and how to update that targeting exercise periodically. We present this methodology in the context of discussions regarding the selection of a targeting methodology in India. In 1992, 1997, and 2002 the Indian government identified households that are

  3. Selecting a Targeting Method to Identify BPL Households in India

    ERIC Educational Resources Information Center

    Alkire, Sabina; Seth, Suman

    2013-01-01

    This paper proposes how to select a methodology to target multidimensionally poor households, and how to update that targeting exercise periodically. We present this methodology in the context of discussions regarding the selection of a targeting methodology in India. In 1992, 1997, and 2002 the Indian government identified households that are…

  4. A stretch of 17 amino acids in the prosaposin C terminus is critical for its binding to sortilin and targeting to lysosomes.

    PubMed

    Yuan, Libin; Morales, Carlos R

    2010-03-01

    Prosaposin, the precursor of four lysosomal cofactors required for the hydrolysis of sphingolipids, is transported to the lysosomes via the alternative receptor, sortilin. In this study, we identified a specific domain of 17 amino acids within the C terminus of prosaposin involved in binding to this sorting receptor. We generated six prosaposin deletion constructs and examined the effect of truncation by coimmunoprecipitation and confocal microscopy. The experiments revealed that the first half of the prosaposin C terminus (aa 524-540), containing a saposin-like motif, was required and necessary to bind sortilin and to transport it to the lysosomes. Based on this result, we introduced twelve site-directed point mutations within the first half of the C terminus. Although the interaction of prosaposin with sortilin was pH dependent, the mutation of hydrophilic amino acids that usually modulate pH-dependent protein interactions did not affect the binding of prosaposin to sortilin. Conversely, a tryptophan (W530) and two cysteines (C528 and C536) were essential for its interaction with sortilin and for its transport to the lysosomes. In conclusion, our investigation demonstrates that a saposin-like motif within the first half of the prosaposin C terminus contains the sortilin recognition site. PMID:19934382

  5. Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting

    PubMed Central

    Ciechanover, Aaron

    2012-01-01

    Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code was transcribed to RNA and translated to proteins, but how proteins were degraded had remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis was largely non-lysosomal, but the mechanisms involved have remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs. PMID:23908826

  6. Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate.

    PubMed

    Kondo, Marcia Y; Gouvea, Iuri E; Okamoto, Débora N; Santos, Jorge A N; Souccar, Caden; Oda, Kohei; Juliano, Luiz; Juliano, Maria A

    2016-02-01

    Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca(2+) and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at FF bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher kcat/KM compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys. PMID:26775801

  7. Saccade target selection in Chinese reading.

    PubMed

    Li, Xingshan; Liu, Pingping; Rayner, Keith

    2015-04-01

    In Chinese reading, there are no spaces to mark the word boundaries, so Chinese readers cannot target their saccades to the center of a word. In this study, we investigated how Chinese readers decide where to move their eyes during reading. To do so, we introduced a variant of the boundary paradigm in which only the target stimulus remained on the screen, displayed at the saccade landing site, after the participant's eyes crossed an invisible boundary. We found that when the saccade target was a word, reaction times in a lexical decision task were shorter when the saccade landing position was closer to the end of that word. These results are consistent with the predictions of a processing-based strategy to determine where to move the eyes. Specifically, this hypothesis assumes that Chinese readers estimate how much information is processed in parafoveal vision and saccade to a location that will carry novel information. PMID:25056006

  8. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    SciTech Connect

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  9. Thyroid lysosomes: the stability of the lysosomal membrane.

    PubMed

    Fouchier, F; Mego, J L; Dang, J; Simon, C

    1983-05-01

    To determine the integrity of lysosomes during their isolation from rat thyroid glands and their subsequent incubation at 37 degrees C, the sedimentability of lysosomal acid phosphatase and thyroglobulin (amount of undisrupted lysosomes) and the latency of sedimentable acid phosphatase (permeability of undisrupted lysosomes) were measured concomitantly. The following results were obtained: (a) During isolation of lysosomes in 0.25 M sucrose medium, mild homogenization of thyroid tissue or cholesterol addition did not modify the amount of undisrupted lysosomes but reduced their permeability. Homogenization in 0.5 M sucrose decreased both the amount and the permeability of undisrupted lysosomes. It also reduced their content of recently iodinated thyroglobulin (Tg). Cholesterol addition had no effect in 0.5 M sucrose medium. (b) During incubations at 37 degrees C of lysosomes, the amount of undisrupted lysosomes decreased progressively while their permeability increased. According to the incubation pH, the permeability of lysosomes prepared in 0.25 M sucrose was either more (pH 8) or less (pH 6) extensively increased than that of lysosomes prepared in 0.5 M sucrose. From these results, we concluded: (a) that isolation and incubation of the thyroid lysosomal fraction induce increased permeability of lysosomes prior to their complete disruption: (b) that recently formed lysosomes (high content of recently iodinated Tg) and aged lysosomes (low content of recently iodinated Tg) differ significantly. Recently formed lysosomes are more permeable, are stabilized by cholesterol and are more extensively disrupted in 0.5 M sucrose medium. During incubations, the permeabilities of these two classes of lysosomes are also differently affected by external pH. PMID:11596502

  10. Tuning target selection algorithms to improve galaxy redshift estimates

    NASA Astrophysics Data System (ADS)

    Hoyle, Ben; Paech, Kerstin; Rau, Markus Michael; Seitz, Stella; Weller, Jochen

    2016-03-01

    We showcase machine learning (ML) inspired target selection algorithms to determine which of all potential targets should be selected first for spectroscopic follow up. Efficient target selection can improve the ML redshift uncertainties as calculated on an independent sample, while requiring less targets to be observed. We compare 7 different ML targeting algorithms with the Sloan Digital Sky Survey (SDSS) target order, and with a random targeting algorithm. The ML inspired algorithms are constructed iteratively by estimating which of the remaining target galaxies will be most difficult for the machine learning methods to accurately estimate redshifts using the previously observed data. This is performed by predicting the expected redshift error and redshift offset (or bias) of all of the remaining target galaxies. We find that the predicted values of bias and error are accurate to better than 10-30% of the true values, even with only limited training sample sizes. We construct a hypothetical follow-up survey and find that some of the ML targeting algorithms are able to obtain the same redshift predictive power with 2-3 times less observing time, as compared to that of the SDSS, or random, target selection algorithms. The reduction in the required follow up resources could allow for a change to the follow-up strategy, for example by obtaining deeper spectroscopy, which could improve ML redshift estimates for deeper test data.

  11. Sexual selection targets cetacean pelvic bones.

    PubMed

    Dines, James P; Otrola-Castillo, Erik; Ralph, Peter; Alas, Jesse; Daley, Timothy; Smith, Andrew D; Dean, Matthew D

    2014-11-01

    Male genitalia evolve rapidly, probably as a result of sexual selection. Whether this pattern extends to the internal infrastructure that influences genital movements remains unknown. Cetaceans (whales and dolphins) offer a unique opportunity to test this hypothesis: since evolving from land-dwelling ancestors, they lost external hind limbs and evolved a highly reduced pelvis that seems to serve no other function except to anchor muscles that maneuver the penis. Here, we create a novel morphometric pipeline to analyze the size and shape evolution of pelvic bones from 130 individuals (29 species) in the context of inferred mating system. We present two main findings: (1) males from species with relatively intense sexual selection (inferred by relative testes size) tend to evolve larger penises and pelvic bones compared to their body length, and (2) pelvic bone shape has diverged more in species pairs that have diverged in inferred mating system. Neither pattern was observed in the anterior-most pair of vertebral ribs, which served as a negative control. This study provides evidence that sexual selection can affect internal anatomy that controls male genitalia. These important functions may explain why cetacean pelvic bones have not been lost through evolutionary time. PMID:25186496

  12. Sexual selection targets cetacean pelvic bones

    PubMed Central

    Dines, J. P.; Otárola-Castillo, E.; Ralph, P.; Alas, J.; Daley, T.; Smith, A. D.; Dean, M. D.

    2014-01-01

    Male genitalia evolve rapidly, probably as a result of sexual selection. Whether this pattern extends to the internal infrastructure that influences genital movements remains unknown. Cetaceans (whales and dolphins) offer a unique opportunity to test this hypothesis: since evolving from land-dwelling ancestors, they lost external hind limbs and evolved a highly reduced pelvis which seems to serve no other function except to anchor muscles that maneuver the penis. Here we create a novel morphometric pipeline to analyze the size and shape evolution of pelvic bones from 130 individuals (29 species) in the context of inferred mating system. We present two main findings: 1) males from species with relatively intense sexual selection (inferred by relative testes size) have evolved relatively large penises and pelvic bones compared to their body size, and 2) pelvic bone shape diverges more quickly in species pairs that have diverged in inferred mating system. Neither pattern was observed in the anterior-most pair of vertebral ribs, which served as a negative control. This study provides evidence that sexual selection can affect internal anatomy that controls male genitalia. These important functions may explain why cetacean pelvic bones have not been lost through evolutionary time. PMID:25186496

  13. PRD125, a potent and selective inhibitor of sterol O-acyltransferase 2 markedly reduces hepatic cholesteryl ester accumulation and improves liver function in lysosomal acid lipase-deficient mice.

    PubMed

    Lopez, Adam M; Chuang, Jen-Chieh; Posey, Kenneth S; Ohshiro, Taichi; Tomoda, Hiroshi; Rudel, Lawrence L; Turley, Stephen D

    2015-11-01

    In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal(-/-) mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal(+/+) littermates (23 versus 1.8 mg, respectively). In Lal(-/-) males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal(-/-) mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management. PMID:26283692

  14. A two-photon fluorescent probe for lysosomal zinc ions.

    PubMed

    Lee, Hyo-Jun; Cho, Chang-Woo; Seo, Hyewon; Singha, Subhankar; Jun, Yong Woong; Lee, Kyung-Ha; Jung, Youngseob; Kim, Kyong-Tai; Park, Seongjun; Bae, Sung Chul; Ahn, Kyo Han

    2015-12-15

    The selective detection of zinc ions in lysosomes over that in cytosol is achieved with a fluorescent probe, which enabled the fluorescence imaging of endogenous zinc ions in lysosomes of NIH 3T3 cells as well as mouse hippocampal tissues by two-photon microscopy under excitation at 900 nm. PMID:26503088

  15. Selectively targeting pain in the trigeminal system

    PubMed Central

    Kim, Hyun Yeong; Kim, Kihwan; Li, Hai Ying; Chung, Gehoon; Park, Chul-Kyu; Kim, Joong Soo; Jung, Sung Jun; Lee, Min Kyung; Ahn, Dong Kuk; Hwang, Se Jin; Kang, Youngnam; Binshtok, Alexander M.; Bean, Bruce P.; Woolf, Clifford J.; Oh, Seog Bae

    2015-01-01

    We tested whether it is possible to selectively block pain signals in the orofacial area by delivering the permanently charged lidocaine derivative QX-314 into nociceptors via TPRV1 channels. We examined the effects of co-applied QX-314 and capsaicin on nociceptive, proprioceptive, and motor function in the rat trigeminal system. QX-314 alone failed to block voltage-gated sodium channel currents (INa) and action potentials (APs) in trigeminal ganglion (TG) neurons. However, co-application of QX-314 and capsaicin blocked INa and APs in TRPV1-positive TG and dental nociceptive neurons, but not in TRPV1-negative TG neurons or in small neurons from TRPV1 knock-out mice. Immunohistochemistry revealed that TRPV1 is not expressed by trigeminal motor and trigeminal mesencephalic neurons. Capsaicin had no effect on rat trigeminal motor and proprioceptive mesencephalic neurons and therefore should not allow QX-314 to enter these cells. Co-application of QX-314 and capsaicin inhibited the jaw-opening reflex evoked by noxious electrical stimulation of the tooth pulp when applied to a sensory but not a motor nerve, and produced long-lasting analgesia in the orofacial area. These data show that selective block of pain signals can be achieved by co-application of QX-314 with TRPV1 agonists. This approach has potential utility in the trigeminal system for treating dental and facial pain. PMID:20236764

  16. General lysosomal hydrolysis can process prorenin accurately.

    PubMed

    Xa, Lucie K; Lacombe, Marie-Jose; Mercure, Chantal; Lazure, Claude; Reudelhuber, Timothy L

    2014-09-01

    Renin, an aspartyl protease that catalyzes the rate-limiting step of the renin-angiotensin system, is first synthesized as an inactive precursor, prorenin. Prorenin is activated by the proteolytic removal of an amino terminal prosegment in the dense granules of the juxtaglomerular (JG) cells of the kidney by one or more proteases whose identity is uncertain but commonly referred to as the prorenin-processing enzyme (PPE). Because several extrarenal tissues secrete only prorenin, we tested the hypothesis that the unique ability of JG cells to produce active renin might be explained by the existence of a PPE whose expression is restricted to JG cells. We found that inducing renin production by the mouse kidney by up to 20-fold was not associated with the concomitant induction of candidate PPEs. Because the renin-containing granules of JG cells also contain several lysosomal hydrolases, we engineered mouse Ren1 prorenin to be targeted to the classical vesicular lysosomes of cultured HEK-293 cells, where it was accurately processed and stored. Furthermore, we found that HEK cell lysosomes hydrolyzed any artificial extensions placed on the protein and that active renin was extraordinarily resistant to proteolytic degradation. Altogether, our results demonstrate that accurate processing of prorenin is not restricted to JG cells but can occur in classical vesicular lysosomes of heterologous cells. The implication is that renin production may not require a specific PPE but rather can be achieved by general hydrolysis in the lysosome-like granules of JG cells. PMID:24965790

  17. Lysosome: regulator of lipid degradation pathways.

    PubMed

    Settembre, Carmine; Ballabio, Andrea

    2014-12-01

    Autophagy is a catabolic pathway that has a fundamental role in the adaptation to fasting and primarily relies on the activity of the endolysosomal system, to which the autophagosome targets substrates for degradation. Recent studies have revealed that the lysosomal-autophagic pathway plays an important part in the early steps of lipid degradation. In this review, we discuss the transcriptional mechanisms underlying co-regulation between lysosome, autophagy, and other steps of lipid catabolism, including the activity of nutrient-sensitive transcription factors (TFs) and of members of the nuclear receptor family. In addition, we discuss how the lysosome acts as a metabolic sensor and orchestrates the transcriptional response to fasting. PMID:25061009

  18. Computational design of nanoparticle drug delivery systems for selective targeting

    NASA Astrophysics Data System (ADS)

    Duncan, Gregg A.; Bevan, Michael A.

    2015-09-01

    Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ?R, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ?L. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues.Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ?R, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ?L. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues. Electronic supplementary information (ESI) available: Movie showing simulation renderings of targeted (?L = 1820/?m2, KD = 120 ?M) nanoparticle selective binding to cancer (?R = 256/?m2) vs. healthy (?R = 64/?m2) cell surfaces. Target membrane proteins have linear color scale depending on binding energy ranging from white when unbound (URL = 0) to red when tightly bound (URL = UM). See DOI: 10.1039/c5nr03691g

  19. UVA Causes Dual Inactivation of Cathepsin B and L Underlying Lysosomal Dysfunction in Human Dermal Fibroblasts

    PubMed Central

    Lamore, Sarah D.; Wondrak, Georg T.

    2013-01-01

    Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display UVA-mimetic effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts. PMID:23603447

  20. Target selection biases from recent experience transfer across effectors.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2016-02-01

    Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions. PMID:26563393

  1. Lysosomal ROS formation.

    PubMed

    Nohl, Hans; Gille, Lars

    2005-01-01

    Ubiquinone is inhomogenously distributed in subcellular biomembranes. Apart from mitochondria, where ubiquinone has bioenergetic and pathophysiological functions, unusually high levels of ubiquinone have also been reported in Golgi vesicles and lysosomes. In lysosomes, the interior differs from other organelles in its low pH value which is important to ensure optimal activity of hydrolytic enzymes. Since redox-cycling of ubiquinone is associated with the acceptance and release of protons, we assumed that ubiquinone is part of a redox chain contributing to unilateral proton distribution. A similar function of ubiquinone was earlier suggested by Crane to operate in Golgi vesicles. Support for the involvement of ubiquinone in a presumed couple of redox carriers came from our observation that almost 70% of total lysosomal ubiquinone was in the divalently reduced state. Further reduction was seen in the presence of external NADH. Analysis of the components involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone revealed the existence of an FAD-containing NADH dehydrogenase. The latter was found to reduce ubiquinone by means of a b-type cytochrome. Proton translocation into the interior was linked to the activity of the novel lysosomal redox chain. Oxygen was found to be the terminal electron acceptor, thereby also regulating acidification of the lysosomal matrix. In contrast to mitochondrial respiration, oxygen was only trivalently reduced giving rise to the release of HO radicals. The role of this novel proton-pumping redox chain and the significance of the associated ROS formation has to be elucidated. PMID:16259787

  2. Selecting asteroids for a targeted spectroscopic survey

    NASA Astrophysics Data System (ADS)

    Oszkiewicz, D. A.; Kwiatkowski, T.; Tomov, T.; Birlan, M.; Geier, S.; Penttil, A.; Poli?ska, M.

    2014-12-01

    Context. Asteroid spectroscopy reflects surface mineralogy. There are a few thousand asteroids whose surfaces have been observed spectrally. Determining their surface properties is important for many practical and scientific applications, such as developing impact deflection strategies or studying the history and evolution of the solar system and planet formation. Aims: The aim of this study is to develop a preselection method that can be used to search for asteroids of any taxonomic complex. The method could then be utilized in multiple applications, such as searching for the missing V-types or looking for primitive asteroids. Methods: We used the Bayes Naive Classifier combined with observations obtained in the course of the Sloan Digital Sky Survey and the Wide-field Infrared Survey Explorer surveys, as well as a database of asteroid phase curves for asteroids with a known taxonomic type. With this new classification method, we selected a number of possible V-type candidates. Some of the candidates were then spectrally observed at the Nordic Optical Telescope and South African Large Telescope. Results: We developed and tested the new preselection method. We found three asteroids in the mid-to-outer main belt that probably have differentiated types. Near-infrared observations are still required to confirm this discovery. As in other studies we found that V-type candidates cluster around the Vesta family and are rare in the mid-to-outer main belt. Conclusions: The new method shows that even largely explored large databases when combined could still be exploited further in, for example, solving the missing dunite problem. Tables 6 and A.1 are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/572/A29

  3. Kinetic evidence that newly-synthesized endogenous lysosome-associated membrane protein-1 (LAMP-1) first transits early endosomes before it is delivered to lysosomes.

    PubMed

    Ebrahim, Roshan; Thilo, Lutz

    2011-05-01

    After de novo synthesis of lysosome-associated membrane proteins (LAMPs), they are sorted in the trans-Golgi network (TGN) for delivery to lysosomes. Opposing views prevail on whether LAMPs are targeted to lysosomes directly, or indirectly via prelysosomal stages of the endocytic pathway, in particular early endosomes. Conflicting evidence is based on kinetic measurements with too limited quantitative data for sufficient temporal and organellar resolution. Using cells of the mouse macrophage cell line, P338D(1), this study presents detailed kinetic data that describe the extent of, and time course for, the appearance of newly-synthesized LAMP-1 in organelles of the endocytic pathway, which had been loaded selectively with horse-radish peroxidase (HRP) by appropriate periods of endocytosis. After a 5-min pulse of metabolic labelling, LAMP-1 was trapped in the respective organelles by HRP-catalyzed crosslinking with membrane-permeable diaminobenzidine (DAB). These kinetic observations provide sufficient quantitative evidence that in P338D(1) cells the bulk of newly-synthesized endogenous LAMP-1 first appeared in early endosomes, before it was delivered to late endosomes and lysosomes about 25 min later. PMID:21457058

  4. TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion.

    PubMed

    Xia, Qin; Wang, Hongfeng; Hao, Zongbing; Fu, Cheng; Hu, Qingsong; Gao, Feng; Ren, Haigang; Chen, Dong; Han, Junhai; Ying, Zheng; Wang, Guanghui

    2016-01-18

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration. PMID:26702100

  5. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-03-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  6. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-01-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  7. Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition.

    PubMed

    Pribil, Patrick A; Haniford, David B

    2003-07-01

    The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA. PMID:12823965

  8. A SIMPLE LIKELIHOOD METHOD FOR QUASAR TARGET SELECTION

    SciTech Connect

    Kirkpatrick, Jessica A.; Schlegel, David J.; Ross, Nicholas P.; Myers, Adam D.; Hennawi, Joseph F.; Sheldon, Erin S.; Schneider, Donald P.; Weaver, Benjamin A.

    2011-12-20

    We present a new method for quasar target selection using photometric fluxes and a Bayesian probabilistic approach. For our purposes, we target quasars using Sloan Digital Sky Survey (SDSS) photometry to a magnitude limit of g = 22. The efficiency and completeness of this technique are measured using the Baryon Oscillation Spectroscopic Survey (BOSS) data taken in 2010. This technique was used for the uniformly selected (CORE) sample of targets in BOSS year-one spectroscopy to be realized in the ninth SDSS data release. When targeting at a density of 40 objects deg{sup -2} (the BOSS quasar targeting density), the efficiency of this technique in recovering z > 2.2 quasars is 40%. The completeness compared to all quasars identified in BOSS data is 65%. This paper also describes possible extensions and improvements for this technique.

  9. Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin.

    PubMed

    Zhou, Xiaolai; Sun, Lirong; Bastos de Oliveira, Francisco; Qi, Xiaoyang; Brown, William J; Smolka, Marcus B; Sun, Ying; Hu, Fenghua

    2015-09-14

    Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations. PMID:26370502

  10. Autophagic/lysosomal dysfunction in Alzheimer’s disease

    PubMed Central

    2013-01-01

    Autophagy serves as the sole catabolic mechanism for degrading organelles and protein aggregates. Increasing evidence implicates autophagic dysfunction in Alzheimer’s disease (AD) and other neurodegenerative diseases associated with protein misprocessing and accumulation. Under physiologic conditions, the autophagic/lysosomal system efficiently recycles organelles and substrate proteins. However, reduced autophagy function leads to the accumulation of proteins and autophagic and lysosomal vesicles. These vesicles contain toxic lysosomal hydrolases as well as the proper cellular machinery to generate amyloid-beta, the major component of AD plaques. Here, we provide an overview of current research focused on the relevance of autophagic/lysosomal dysfunction in AD pathogenesis as well as potential therapeutic targets aimed at restoring autophagic/lysosomal pathway function. PMID:24171818

  11. Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

    PubMed Central

    Zhou, Xiaolai; Sun, Lirong; Bastos de Oliveira, Francisco; Qi, Xiaoyang; Brown, William J.; Smolka, Marcus B.; Sun, Ying

    2015-01-01

    Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations. PMID:26370502

  12. Spastic paraplegia proteins spastizin and spatacsin mediate autophagic lysosome reformation

    PubMed Central

    Chang, Jaerak; Lee, Seongju; Blackstone, Craig

    2014-01-01

    Autophagy allows cells to adapt to changes in their environment by coordinating the degradation and recycling of cellular components and organelles to maintain homeostasis. Lysosomes are organelles critical for terminating autophagy via their fusion with mature autophagosomes to generate autolysosomes that degrade autophagic materials; therefore, maintenance of the lysosomal population is essential for autophagy-dependent cellular clearance. Here, we have demonstrated that the two most common autosomal recessive hereditary spastic paraplegia gene products, the SPG15 protein spastizin and the SPG11 protein spatacsin, are pivotal for autophagic lysosome reformation (ALR), a pathway that generates new lysosomes. Lysosomal targeting of spastizin required an intact FYVE domain, which binds phosphatidylinositol 3-phosphate. Loss of spastizin or spatacsin resulted in depletion of free lysosomes, which are competent to fuse with autophagosomes, and an accumulation of autolysosomes, reflecting a failure in ALR. Moreover, spastizin and spatacsin were essential components for the initiation of lysosomal tubulation. Together, these results link dysfunction of the autophagy/lysosomal biogenesis machinery to neurodegeneration. PMID:25365221

  13. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    PubMed Central

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  14. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    PubMed

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonalves de Oliveira; Rigato, Paula Ordonhez; Gonalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  15. Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells

    PubMed Central

    Coen, Katrijn; Flannagan, Ronald S.; Baron, Szilvia; Carraro-Lacroix, Luciene R.; Wang, Dong; Vermeire, Wendy; Michiels, Christine; Munck, Sebastian; Baert, Veerle; Sugita, Shuzo; Wuytack, Frank; Hiesinger, Peter Robin; Grinstein, Sergio

    2012-01-01

    Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: http://dx.doi.org/10.1016/j.cell.2010.05.008) attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN−/− cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN−/− cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency. PMID:22753898

  16. Target Selection for the LBTI Exozodi Key Science Program

    NASA Astrophysics Data System (ADS)

    Weinberger, Alycia J.; Bryden, Geoff; Kennedy, Grant M.; Roberge, Aki; Defrère, Denis; Hinz, Philip M.; Millan-Gabet, Rafael; Rieke, George; Bailey, Vanessa P.; Danchi, William C.; Haniff, Chris; Mennesson, Bertrand; Serabyn, Eugene; Skemer, Andrew J.; Stapelfeldt, Karl R.; Wyatt, Mark C.

    2015-02-01

    The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer will survey nearby stars for faint emission arising from ~300 K dust (exozodiacal dust), and aims to determine the exozodiacal dust luminosity function. HOSTS results will enable planning for future space telescopes aimed at direct spectroscopy of habitable zone terrestrial planets, as well as greater understanding of the evolution of exozodiacal disks and planetary systems. We lay out here the considerations that lead to the final HOSTS target list. Our target selection strategy maximizes the ability of the survey to constrain the exozodi luminosity function by selecting a combination of stars selected for suitability as targets of future missions and as sensitive exozodi probes. With a survey of approximately 50 stars, we show that HOSTS can enable an understanding of the statistical distribution of warm dust around various types of stars and is robust to the effects of varying levels of survey sensitivity induced by weather conditions.

  17. HECT-type Ubiquitin Ligase ITCH Targets Lysosomal-associated Protein Multispanning Transmembrane 5 (LAPTM5) and Prevents LAPTM5-mediated Cell Death*

    PubMed Central

    Ishihara, Takaya; Inoue, Jun; Kozaki, Ken-ichi; Imoto, Issei; Inazawa, Johji

    2011-01-01

    LAPTM5 (lysosomal-associated protein multispanning transmembrane 5) is a membrane protein on the intracellular vesicles. We have previously demonstrated that the accumulation of LAPTM5-positive vesicles was closely associated with the programmed cell death occurring during the spontaneous regression of neuroblastomas. Although the accumulation of LAPTM5 protein might occur at the post-translational level, the molecular mechanism has been unclear. Here, we found that the level of LAPTM5 protein is regulated negatively by the degradation through ubiquitination by ITCH, an E3 ubiquitin ligase. ITCH directly binds to the PPxY motif of LAPTM5 via its WW domains and promotes ubiquitination through a HECT-type ligase domain. Overexpression of ITCH led to the degradation of LAPTM5 protein, and conversely, knockdown of ITCH by siRNA resulted in the stabilization of LAPTM5 protein. In addition, the inhibition of ITCH enhanced the cell death occurred by accumulation of LAPTM5 in neuroblastoma cells. These findings suggest that LAPTM5 is a novel substrate in terms of degradation by the ubiquitin ligase ITCH, and this system might act as a negative regulator in the spontaneous regression of neuroblastomas by preventing LAPTM5-mediated cell death. PMID:22009753

  18. Ethambutol neutralizes lysosomes and causes lysosomal zinc accumulation.

    PubMed

    Yamada, Daisuke; Saiki, Shinji; Furuya, Norihiko; Ishikawa, Kei-Ichi; Imamichi, Yoko; Kambe, Taiho; Fujimura, Tsutomu; Ueno, Takashi; Koike, Masato; Sumiyoshi, Katsuhiko; Hattori, Nobutaka

    2016-02-26

    Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn(2+) accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn(2+) accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism. PMID:26851368

  19. Lysosomes: Regulators of autophagy in the retinal pigmented epithelium.

    PubMed

    Sinha, Debasish; Valapala, Mallika; Shang, Peng; Hose, Stacey; Grebe, Rhonda; Lutty, Gerard A; Zigler, J Samuel; Kaarniranta, Kai; Handa, James T

    2016-03-01

    The retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to its very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. This signaling pathway is becoming a target for therapeutic intervention in diseases, including age-related macular degeneration (AMD), where lysosomal function is defective. In addition, our laboratory has been studying animal models in which the gene (Cryba1) for ?A3/A1-crystallin is deficient. These animals exhibit impaired lysosomal clearance in the RPE and pathological signs that are similar to some of those seen in AMD patients. The data demonstrate that ?A3/A1-crystallin localizes to lysosomes in the RPE and that it is a binding partner of V-ATPase, the proton pump that acidifies the lysosomal lumen. This suggests that ?A3/A1-crystallin may also be a potential target for therapeutic intervention in AMD. In this review, we focus on effector molecules that impact the lysosomal-autophagic pathway in RPE cells. PMID:26321509

  20. Feature Extraction and Selection Strategies for Automated Target Recognition

    NASA Technical Reports Server (NTRS)

    Greene, W. Nicholas; Zhang, Yuhan; Lu, Thomas T.; Chao, Tien-Hsin

    2010-01-01

    Several feature extraction and selection methods for an existing automatic target recognition (ATR) system using JPLs Grayscale Optical Correlator (GOC) and Optimal Trade-Off Maximum Average Correlation Height (OT-MACH) filter were tested using MATLAB. The ATR system is composed of three stages: a cursory region of-interest (ROI) search using the GOC and OT-MACH filter, a feature extraction and selection stage, and a final classification stage. Feature extraction and selection concerns transforming potential target data into more useful forms as well as selecting important subsets of that data which may aide in detection and classification. The strategies tested were built around two popular extraction methods: Principal Component Analysis (PCA) and Independent Component Analysis (ICA). Performance was measured based on the classification accuracy and free-response receiver operating characteristic (FROC) output of a support vector machine(SVM) and a neural net (NN) classifier.

  1. Autophagic-lysosomal dysregulation downstream of cathepsin B inactivation in human skin fibroblasts exposed to UVA

    PubMed Central

    Lamore, Sarah D.; Wondrak, Georg T.

    2014-01-01

    Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J/cm2, twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and α-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, α-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage. PMID:21773629

  2. Target SNP selection in complex disease association studies

    PubMed Central

    Wjst, Matthias

    2004-01-01

    Background The massive amount of SNP data stored at public internet sites provides unprecedented access to human genetic variation. Selecting target SNP for disease-gene association studies is currently done more or less randomly as decision rules for the selection of functional relevant SNPs are not available. Results We implemented a computational pipeline that retrieves the genomic sequence of target genes, collects information about sequence variation and selects functional motifs containing SNPs. Motifs being considered are gene promoter, exon-intron structure, AU-rich mRNA elements, transcription factor binding motifs, cryptic and enhancer splice sites together with expression in target tissue. As a case study, 396 genes on chromosome 6p21 in the extended HLA region were selected that contributed nearly 20,000 SNPs. By computer annotation ~2,500 SNPs in functional motifs could be identified. Most of these SNPs are disrupting transcription factor binding sites but only those introducing new sites had a significant depressing effect on SNP allele frequency. Other decision rules concern position within motifs, the validity of SNP database entries, the unique occurrence in the genome and conserved sequence context in other mammalian genomes. Conclusion Only 10% of all gene-based SNPs have sequence-predicted functional relevance making them a primary target for genotyping in association studies. PMID:15248903

  3. Perceptual task induces saccadic adaptation by target selection.

    PubMed

    Schtz, Alexander C; Souto, David

    2015-01-01

    Adaptation of saccades can be induced by different error signals, such as retinal position errors, prediction errors, or reinforcement learning. Recently, we showed that a shift in the spatial goal of a perceptual task can induce saccadic adaptation, in the absence of a bottom-up position error. Here, we investigated whether this top-down effect is mediated by the visibility of the task-relevant object, by reinforcement due to the feedback about the perceptual judgment or by a target selection mechanism. Participants were asked to discriminate visual stimuli arranged in a vertical compound. To induce adaptation, the discrimination target was presented at eccentric locations in the compound. In the first experiment, we compared adaptation with an easy and difficult discrimination. In the second experiment, we compared adaptation when feedback about the perceptual task was valid and when feedback was provided but was unrelated to performance. In the third experiment, we compared adaptation with instructions to fixate one of the elements in the compound-target selection-to the perceptual task condition-target selection and discrimination. To control for a bottom-up stimulus effect, we ran a fourth experiment in which the only instruction was to look at the compound. The saccade amplitude data were fitted by a two-state model distinguishing between an immediate and a gradual error correction process. We replicated our finding that a perceptual task can drive adaptation of saccades. Adaptation showed no effect of feedback reliability, nor an effect of the perceptual task beyond target selection. Adaptation was induced by a top-down signal since it was absent when there was no target selection instruction and no perceptual task. The immediate error correction was larger for the difficult than for the easy condition, suggesting that task difficulty affects mainly voluntary saccade targeting. In addition, the repetition of experiments one week later increased the magnitude of the gradual error correction. The results dissociate two distinct components of adaptation: an immediate and a gradual error correction. We conclude that perceptual-task induced adaptation is most likely due to top-down target selection within a larger object. PMID:26539095

  4. Highly Stable and Sensitive Fluorescent Probes (LysoProbes) for Lysosomal Labeling and Tracking

    PubMed Central

    Yapici, Nazmiye B.; Bi, Yue; Li, Pengfei; Chen, Xin; Yan, Xin; Mandalapu, Srinivas Rao; Faucett, Megan; Jockusch, Steffen; Ju, Jingfang; Gibson, K. Michael; Pavan, William J.; Bi, Lanrong

    2015-01-01

    We report the design, synthesis and application of several new fluorescent probes (LysoProbes I-VI) that facilitate lysosomal pH monitoring and characterization of lysosome-dependent apoptosis. LysoProbes are superior to commercially available lysosome markers since the fluorescent signals are both stable and highly selective, and they will aid in characterization of lysosome morphology and trafficking. We predict that labeling of cancer cells and solid tumor tissues with LysoProbes will provide an important new tool for monitoring the role of lysosome trafficking in cancer invasion and metastasis. PMID:25715948

  5. Selective screening for lysosomal storage diseases with dried blood spots collected on filter paper in 4,700 high-risk colombian subjects.

    PubMed

    Uribe, Alfredo; Giugliani, Roberto

    2013-01-01

    Lysosomal storage disorders (LSDs) are a very heterogeneous group of hereditary disorders. The diagnostic process usually involves complex sampling, processing, testing, and validation procedures, performed by specialized laboratories only, which causes great limitations in reaching a diagnosis for patients affected by these diseases.There are few studies about LSDs in Colombia. The diagnostic limitations often make medical practitioners disregard the possibility of these disorders while diagnosing their patients. The current study documents the results of a 7-year screening in high-risk patients, aimed to detect LSDs using dried blood spots (DBS) collected on filter paper, with a micromethodology that facilitates diagnosis even with a large number of samples.The activities of ?-galactosidase A, ? glucosidase, ?-L-iduronidase, arylsulfatase B, ?-galactosidase, ?-glucosidase, total hexosaminidase, iduronate sulfatase, and chitotriosidase were analyzed in high-risk patients for lysosomal disease. The catalytic activity was evaluated with fluorometric micromethods using artificial substrates marked with 4-methylumbelliferone.The reference values for a control population were established for the enzymes listed above, and 242 patients were found to have an enzyme deficiency, guiding to the following diagnoses: Fabry disease (n = 31), Pompe disease (n = 16), Hurler Syndrome (n = 15), Maroteaux-Lamy Syndrome (n = 34), GM1 Gangliosidosis (n = 10), Morquio B (n = 1), Gaucher disease (n = 101), Sandhoff disease (n = 1), Mucolipidosis (n = 2), and Hunter Syndrome (n = 31). In conclusion, this protocol provides a comprehensive diagnostic approach which could be carried out in Colombia and made it available to medical services spread around the country, enabling the identification of a large number of patients affected by LSDs, which could potentially benefit from the therapeutic tools already available for many of these diseases. PMID:23609959

  6. Perceptual task induces saccadic adaptation by target selection

    PubMed Central

    Schütz, Alexander C.; Souto, David

    2015-01-01

    Adaptation of saccades can be induced by different error signals, such as retinal position errors, prediction errors, or reinforcement learning. Recently, we showed that a shift in the spatial goal of a perceptual task can induce saccadic adaptation, in the absence of a bottom-up position error. Here, we investigated whether this top-down effect is mediated by the visibility of the task-relevant object, by reinforcement due to the feedback about the perceptual judgment or by a target selection mechanism. Participants were asked to discriminate visual stimuli arranged in a vertical compound. To induce adaptation, the discrimination target was presented at eccentric locations in the compound. In the first experiment, we compared adaptation with an easy and difficult discrimination. In the second experiment, we compared adaptation when feedback about the perceptual task was valid and when feedback was provided but was unrelated to performance. In the third experiment, we compared adaptation with instructions to fixate one of the elements in the compound—target selection—to the perceptual task condition—target selection and discrimination. To control for a bottom-up stimulus effect, we ran a fourth experiment in which the only instruction was to look at the compound. The saccade amplitude data were fitted by a two-state model distinguishing between an immediate and a gradual error correction process. We replicated our finding that a perceptual task can drive adaptation of saccades. Adaptation showed no effect of feedback reliability, nor an effect of the perceptual task beyond target selection. Adaptation was induced by a top-down signal since it was absent when there was no target selection instruction and no perceptual task. The immediate error correction was larger for the difficult than for the easy condition, suggesting that task difficulty affects mainly voluntary saccade targeting. In addition, the repetition of experiments one week later increased the magnitude of the gradual error correction. The results dissociate two distinct components of adaptation: an immediate and a gradual error correction. We conclude that perceptual-task induced adaptation is most likely due to top-down target selection within a larger object. PMID:26539095

  7. Integrative analysis to select cancer candidate biomarkers to targeted validation

    PubMed Central

    Heberle, Henry; Domingues, Romênia R.; Granato, Daniela C.; Yokoo, Sami; Canevarolo, Rafael R.; Winck, Flavia V.; Ribeiro, Ana Carolina P.; Brandão, Thaís Bianca; Filgueiras, Paulo R.; Cruz, Karen S. P.; Barbuto, José Alexandre; Poppi, Ronei J.; Minghim, Rosane; Telles, Guilherme P.; Fonseca, Felipe Paiva; Fox, Jay W.; Santos-Silva, Alan R.; Coletta, Ricardo D.; Sherman, Nicholas E.; Paes Leme, Adriana F.

    2015-01-01

    Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS. PMID:26540631

  8. Integrative analysis to select cancer candidate biomarkers to targeted validation.

    PubMed

    Kawahara, Rebeca; Meirelles, Gabriela V; Heberle, Henry; Domingues, Romênia R; Granato, Daniela C; Yokoo, Sami; Canevarolo, Rafael R; Winck, Flavia V; Ribeiro, Ana Carolina P; Brandão, Thaís Bianca; Filgueiras, Paulo R; Cruz, Karen S P; Barbuto, José Alexandre; Poppi, Ronei J; Minghim, Rosane; Telles, Guilherme P; Fonseca, Felipe Paiva; Fox, Jay W; Santos-Silva, Alan R; Coletta, Ricardo D; Sherman, Nicholas E; Paes Leme, Adriana F

    2015-12-22

    Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS. PMID:26540631

  9. Target Selection for the SDSS-III MARVELS Survey

    NASA Astrophysics Data System (ADS)

    Paegert, Martin; Stassun, Keivan G.; De Lee, Nathan; Pepper, Joshua; Fleming, Scott W.; Sivarani, Thirupathi; Mahadevan, Suvrath; Mack, Claude E., III; Dhital, Saurav; Hebb, Leslie; Ge, Jian

    2015-06-01

    We present the target selection process for the Multi-object APO Radial Velocity Exoplanets Large-area Survey (MARVELS), which is part of the Sloan Digital Sky Survey (SDSS) III. MARVELS is a medium-resolution (R ? 11,000) multi-fiber spectrograph capable of obtaining radial velocities for 60 objects at a time in order to find brown dwarfs and giant planets. The survey was configured to target dwarf stars with effective temperatures approximately between 4500 and 6250 K. For the first 2 years MARVELS relied on low-resolution spectroscopic pre-observations to estimate the effective temperature and log (g) for candidate stars and then selected suitable dwarf stars from this pool. Ultimately, the pre-observation spectra proved ineffective at filtering out giant stars; many giants were incorrectly classified as dwarfs, resulting in a giant contamination rate of ?30% for the first phase of the MARVELS survey. Thereafter, the survey instead applied a reduced proper motion cut to eliminate giants and used the Infrared Flux Method to estimate effective temperatures, using only extant photmetric and proper-motion catalog information. The target selection method introduced here may be useful for other surveys that need to rely on extant catalog data for selection of specific stellar populations.

  10. Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics

    PubMed Central

    Kiyonami, Reiko; Schoen, Alan; Prakash, Amol; Peterman, Scott; Zabrouskov, Vlad; Picotti, Paola; Aebersold, Ruedi; Huhmer, Andreas; Domon, Bruno

    2011-01-01

    Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies. PMID:20664071

  11. Highly selective luminescent nanostructures for mitochondrial imaging and targeting.

    PubMed

    Fanizza, E; Iacobazzi, R M; Laquintana, V; Valente, G; Caliandro, G; Striccoli, M; Agostiano, A; Cutrignelli, A; Lopedota, A; Curri, M L; Franco, M; Depalo, N; Denora, N

    2016-02-14

    Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a]pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Sub-cellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents. PMID:26763470

  12. Neuroinflammatory paradigms in lysosomal storage diseases

    PubMed Central

    Bosch, Megan E.; Kielian, Tammy

    2015-01-01

    Lysosomal storage diseases (LSDs) include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss. PMID:26578874

  13. Histone target selection within chromatin: an exemplary case of teamwork.

    PubMed

    Lalonde, Marie-Eve; Cheng, Xue; Ct, Jacques

    2014-05-15

    Histone modifiers like acetyltransferases, methyltransferases, and demethylases are critical regulators of most DNA-based nuclear processes, de facto controlling cell cycle progression and cell fate. These enzymes perform very precise post-translational modifications on specific histone residues, which in turn are recognized by different effector modules/proteins. We now have a better understanding of how these enzymes exhibit such specificity. As they often reside in multisubunit complexes, they use associated factors to target their substrates within chromatin structure and select specific histone mark-bearing nucleosomes. In this review, we cover the current understanding of how histone modifiers select their histone targets. We also explain how different experimental approaches can lead to conflicting results about the histone specificity and function of these enzymes. PMID:24831698

  14. Autophagy, lipophagy and lysosomal lipid storage disorders.

    PubMed

    Ward, Carl; Martinez-Lopez, Nuria; Otten, Elsje G; Carroll, Bernadette; Maetzel, Dorothea; Singh, Rajat; Sarkar, Sovan; Korolchuk, Viktor I

    2016-04-01

    Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders. PMID:26778751

  15. Identification and Characterization of Pharmacological Chaperones to Correct Enzyme Deficiencies in Lysosomal Storage Disorders

    PubMed Central

    Khanna, Richie; Powe, Allan C.; Boyd, Robert; Lee, Gary; Flanagan, John J.; Benjamin, Elfrida R.

    2011-01-01

    Abstract Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed. PMID:21612550

  16. Selective targeting of Mycobacterium smegmatis with trehalose-functionalized nanoparticles.

    PubMed

    Jayawardana, Kalana W; Jayawardena, H Surangi N; Wijesundera, Samurdhi A; De Zoysa, Thareendra; Sundhoro, Madanodaya; Yan, Mingdi

    2015-08-01

    Silica and iron oxide nanoparticles with sizes ranging from 6 to 40 nm were functionalized with trehalose. The trehalose-conjugated nanoparticles showed strong interactions with Mycobacterium smegmatis (M. smegmatis) and minimal interactions with macrophage (RAW 264.7) or A549 cells. In addition, trehalose-conjugated silica nanoparticles selectively interacted with M. smegmatis on M. smegmatis-treated A549 cells, demonstrating high potential of trehalose in developing targeted therapy for treating mycobacterial infection. PMID:26121049

  17. Neuroactive insecticides: targets, selectivity, resistance, and secondary effects.

    PubMed

    Casida, John E; Durkin, Kathleen A

    2013-01-01

    Neuroactive insecticides are the principal means of protecting crops, people, livestock, and pets from pest insect attack and disease transmission. Currently, the four major nerve targets are acetylcholinesterase for organophosphates and methylcarbamates, the nicotinic acetylcholine receptor for neonicotinoids, the ?-aminobutyric acid receptor/chloride channel for polychlorocyclohexanes and fiproles, and the voltage-gated sodium channel for pyrethroids and dichlorodiphenyltrichloroethane. Species selectivity and acquired resistance are attributable in part to structural differences in binding subsites, receptor subunit interfaces, or transmembrane regions. Additional targets are sites in the sodium channel (indoxacarb and metaflumizone), the glutamate-gated chloride channel (avermectins), the octopamine receptor (amitraz metabolite), and the calcium-activated calcium channel (diamides). Secondary toxic effects in mammals from off-target serine hydrolase inhibition include organophosphate-induced delayed neuropathy and disruption of the cannabinoid system. Possible associations between pesticides and Parkinson's and Alzheimer's diseases are proposed but not established based on epidemiological observations and mechanistic considerations. PMID:23317040

  18. Target Selection for the LBTI Exozodi Key Science Program

    NASA Technical Reports Server (NTRS)

    Weinberger, Alycia J.; Bryden, Geoff; Kennedy, Grant M.; Roberge, Aki; Defrere, Denis; Hinz, Philip M.; Millan-Gabet, Rafael; Rieke, George; Bailey, Vanessa P.; Danchi, William C.; Haniff, Chris; Mennesson, Bertrand; Serabyn, Eugne; Skemer, Andrew J.; Stapelfeldt, Karl R.; Wyatt, Mark C.

    2015-01-01

    The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer will survey nearby stars for faint emission arising from approximately 300K dust (exozodiacal dust), and aims to determine the exozodiacal dust luminosity function. HOSTS results will enable planning for future space telescopes aimed at direct spectroscopy of habitable zone terrestrial planets, as well as greater understanding of the evolution of exozodiacal disks and planetary systems. We lay out here the considerations that lead to the final HOSTS target list. Our target selection strategy maximizes the ability of the survey to constrain the exozodi luminosity function by selecting a combination of stars selected for suitability as targets of future missions and as sensitive exozodi probes. With a survey of approximately 50 stars, we show that HOSTS can enable an understanding of the statistical distribution of warm dust around various types of stars and is robust to the effects of varying levels of survey sensitivity induced by weather conditions.

  19. TARGET SELECTION FOR THE LBTI EXOZODI KEY SCIENCE PROGRAM

    SciTech Connect

    Weinberger, Alycia J.; Bryden, Geoff; Mennesson, Bertrand; Serabyn, Eugene; Kennedy, Grant M.; Wyatt, Mark C.; Roberge, Aki; Danchi, William C.; Stapelfeldt, Karl R.; Defrère, Denis; Hinz, Philip M.; Rieke, George; Bailey, Vanessa P.; Skemer, Andrew J.; Millan-Gabet, Rafael; Haniff, Chris

    2015-02-01

    The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer will survey nearby stars for faint emission arising from ∼300 K dust (exozodiacal dust), and aims to determine the exozodiacal dust luminosity function. HOSTS results will enable planning for future space telescopes aimed at direct spectroscopy of habitable zone terrestrial planets, as well as greater understanding of the evolution of exozodiacal disks and planetary systems. We lay out here the considerations that lead to the final HOSTS target list. Our target selection strategy maximizes the ability of the survey to constrain the exozodi luminosity function by selecting a combination of stars selected for suitability as targets of future missions and as sensitive exozodi probes. With a survey of approximately 50 stars, we show that HOSTS can enable an understanding of the statistical distribution of warm dust around various types of stars and is robust to the effects of varying levels of survey sensitivity induced by weather conditions.

  20. PDT: loss of autophagic cytoprotection after lysosomal photodamage

    NASA Astrophysics Data System (ADS)

    Kessel, David; Price, Michael

    2012-02-01

    Photodynamic therapy is known to evoke both autophagy and apoptosis. Apoptosis is an irreversible death pathway while autophagy can serve a cytoprotective function. In this study, we examined two photosensitizing agents that target lysosomes, although they differ in the reactive oxygen species (ROS) formed during irradiation. With both agents, the 'shoulder' on the PDT dose-response curve was substantially attenuated, consistent with loss of a cytoprotective pathway. In contrast, this 'shoulder' is commonly observed when PDT targets mitochondria or the ER. We propose that lysosomal targets may offer the possibility of promoting PDT efficacy by eliminating a potentially protective pathway.

  1. Brevinin-2R1 semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway

    PubMed Central

    Ghavami, Saeid; Asoodeh, Ahmad; Klonisch, Thomas; Halayko, Andrew J; Kadkhoda, Kamran; Kroczak, Tadeusz J; Gibson, Spencer B; Booy, Evan P; Naderi-Manesh, Hossein; Los, Marek

    2008-01-01

    Abstract Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (ΔTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (ΔΨm), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death. PMID:18494941

  2. A Deterministic Approach to Active Debris Removal Target Selection

    NASA Astrophysics Data System (ADS)

    Lidtke, A.; Lewis, H.; Armellin, R.

    2014-09-01

    Many decisions, with widespread economic, political and legal consequences, are being considered based on space debris simulations that show that Active Debris Removal (ADR) may be necessary as the concerns about the sustainability of spaceflight are increasing. The debris environment predictions are based on low-accuracy ephemerides and propagators. This raises doubts about the accuracy of those prognoses themselves but also the potential ADR target-lists that are produced. Target selection is considered highly important as removal of many objects will increase the overall mission cost. Selecting the most-likely candidates as soon as possible would be desirable as it would enable accurate mission design and allow thorough evaluation of in-orbit validations, which are likely to occur in the near-future, before any large investments are made and implementations realized. One of the primary factors that should be used in ADR target selection is the accumulated collision probability of every object. A conjunction detection algorithm, based on the smart sieve method, has been developed. Another algorithm is then applied to the found conjunctions to compute the maximum and true probabilities of collisions taking place. The entire framework has been verified against the Conjunction Analysis Tools in AGIs Systems Toolkit and relative probability error smaller than 1.5% has been achieved in the final maximum collision probability. Two target-lists are produced based on the ranking of the objects according to the probability they will take part in any collision over the simulated time window. These probabilities are computed using the maximum probability approach, that is time-invariant, and estimates of the true collision probability that were computed with covariance information. The top-priority targets are compared, and the impacts of the data accuracy and its decay are highlighted. General conclusions regarding the importance of Space Surveillance and Tracking for the purpose of ADR are also drawn and a deterministic method for ADR target selection, which could reduce the number of ADR missions to be performed, is proposed.

  3. The SAMI Galaxy Survey: instrument specification and target selection

    NASA Astrophysics Data System (ADS)

    Bryant, J. J.; Owers, M. S.; Robotham, A. S. G.; Croom, S. M.; Driver, S. P.; Drinkwater, M. J.; Lorente, N. P. F.; Cortese, L.; Scott, N.; Colless, M.; Schaefer, A.; Taylor, E. N.; Konstantopoulos, I. S.; Allen, J. T.; Baldry, I.; Barnes, L.; Bauer, A. E.; Bland-Hawthorn, J.; Bloom, J. V.; Brooks, A. M.; Brough, S.; Cecil, G.; Couch, W.; Croton, D.; Davies, R.; Ellis, S.; Fogarty, L. M. R.; Foster, C.; Glazebrook, K.; Goodwin, M.; Green, A.; Gunawardhana, M. L.; Hampton, E.; Ho, I.-T.; Hopkins, A. M.; Kewley, L.; Lawrence, J. S.; Leon-Saval, S. G.; Leslie, S.; McElroy, R.; Lewis, G.; Liske, J.; Lpez-Snchez, . R.; Mahajan, S.; Medling, A. M.; Metcalfe, N.; Meyer, M.; Mould, J.; Obreschkow, D.; O'Toole, S.; Pracy, M.; Richards, S. N.; Shanks, T.; Sharp, R.; Sweet, S. M.; Thomas, A. D.; Tonini, C.; Walcher, C. J.

    2015-03-01

    The SAMI Galaxy Survey will observe 3400 galaxies with the Sydney-AAO Multi-object Integral-field spectrograph (SAMI) on the Anglo-Australian Telescope in a 3-yr survey which began in 2013. We present the throughput of the SAMI system, the science basis and specifications for the target selection, the survey observation plan and the combined properties of the selected galaxies. The survey includes four volume-limited galaxy samples based on cuts in a proxy for stellar mass, along with low-stellar-mass dwarf galaxies all selected from the Galaxy And Mass Assembly (GAMA) survey. The GAMA regions were selected because of the vast array of ancillary data available, including ultraviolet through to radio bands. These fields are on the celestial equator at 9, 12 and 14.5 h, and cover a total of 144 deg2 (in GAMA-I). Higher density environments are also included with the addition of eight clusters. The clusters have spectroscopy from 2-degree Field Galaxy Redshift Survey (2dFGRS) and Sloan Digital Sky Survey (SDSS) and photometry in regions covered by the SDSS and/or VLT Survey Telescope/ATLAS. The aim is to cover a broad range in stellar mass and environment, and therefore the primary survey targets cover redshifts 0.004 < z < 0.095, magnitudes rpet < 19.4, stellar masses 107-1012 M?, and environments from isolated field galaxies through groups to clusters of 1015 M?.

  4. Selection of molecular targets for drug development against trypanosomatids.

    PubMed

    Smirlis, Despina; Soares, Milena Botelho Pereira

    2014-01-01

    Trypanosomatid parasites are a group of flagellated protozoa that includes the genera Leishmania and Trypanosoma, which are the causative agents of diseases (leishmaniases, sleeping sickness and Chagas disease) that cause considerable morbidity and mortality, affecting more than 27 million people worldwide. Today no effective vaccines for the prevention of these diseases exist, whereas current chemotherapy is ineffective, mainly due to toxic side effects of current drugs and to the emergence of drug resistance and lack of cost effectiveness. For these reasons, rational drug design and the search of good candidate drug targets is of prime importance. The search for drug targets requires a multidisciplinary approach. To this end, the completion of the genome project of many trypanosomatid species gives a vast amount of new information that can be exploited for the identification of good drug candidates with a prediction of "druggability" and divergence from mammalian host proteins. In addition, an important aspect in the search for good drug targets is the "target identification" and evaluation in a biological pathway, as well as the essentiality of the gene in the mammalian stage of the parasite, which is provided by basic research and genetic and proteomic approaches. In this chapter we will discuss how these bioinformatic tools and experimental evaluations can be integrated for the selection of candidate drug targets, and give examples of metabolic and signaling pathways in the parasitic protozoa that can be exploited for rational drug design. PMID:24264240

  5. Autophagy in lysosomal storage disorders

    PubMed Central

    Lieberman, Andrew P.; Puertollano, Rosa; Raben, Nina; Slaugenhaupt, Susan; Walkley, Steven U.; Ballabio, Andrea

    2012-01-01

    Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as “autophagy disorders.” PMID:22647656

  6. Context-dependent sequential effects of target selection for action.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2013-01-01

    Humans exhibit variation in behavior from moment to moment even when performing a simple, repetitive task. Errors are typically followed by cautious responses, minimizing subsequent distractor interference. However, less is known about how variation in the execution of an ultimately correct response affects subsequent behavior. We asked participants to reach toward a uniquely colored target presented among distractors and created two categories to describe participants' responses in correct trials based on analyses of movement trajectories; partial errors referred to trials in which observers initially selected a nontarget for action before redirecting the movement and accurately pointing to the target, and direct movements referred to trials in which the target was directly selected for action. We found that latency to initiate a hand movement was shorter in trials following partial errors compared to trials following direct movements. Furthermore, when the target and distractor colors were repeated, movement time and reach movement curvature toward distractors were greater following partial errors compared to direct movements. Finally, when the colors were repeated, partial errors were more frequent than direct movements following partial-error trials, and direct movements were more frequent following direct-movement trials. The dependence of these latter effects on repeated-task context indicates the involvement of higher-level cognitive mechanisms in an integrated attention-action system in which execution of a partial-error or direct-movement response affects memory representations that bias performance in subsequent trials. Altogether, these results demonstrate that whether a nontarget is selected for action or not has a measurable impact on subsequent behavior. PMID:23847303

  7. Highly selective luminescent nanostructures for mitochondrial imaging and targeting

    NASA Astrophysics Data System (ADS)

    Fanizza, E.; Iacobazzi, R. M.; Laquintana, V.; Valente, G.; Caliandro, G.; Striccoli, M.; Agostiano, A.; Cutrignelli, A.; Lopedota, A.; Curri, M. L.; Franco, M.; Depalo, N.; Denora, N.

    2016-02-01

    Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a]pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Sub-cellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents.Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a]pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Sub-cellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents. Electronic supplementary information (ESI) available: Additional TEM micrographs, fluorescence and UV-Vis absorbance spectra of silica coated QD nanoparticles and TSPO ligand. See DOI: 10.1039/c5nr08139d

  8. Dynamic interactions between visual working memory and saccade target selection

    PubMed Central

    Schneegans, Sebastian; Spencer, John P.; Schöner, Gregor; Hwang, Seongmin; Hollingworth, Andrew

    2014-01-01

    Recent psychophysical experiments have shown that working memory for visual surface features interacts with saccadic motor planning, even in tasks where the saccade target is unambiguously specified by spatial cues. Specifically, a match between a memorized color and the color of either the designated target or a distractor stimulus influences saccade target selection, saccade amplitudes, and latencies in a systematic fashion. To elucidate these effects, we present a dynamic neural field model in combination with new experimental data. The model captures the neural processes underlying visual perception, working memory, and saccade planning relevant to the psychophysical experiment. It consists of a low-level visual sensory representation that interacts with two separate pathways: a spatial pathway implementing spatial attention and saccade generation, and a surface feature pathway implementing color working memory and feature attention. Due to bidirectional coupling between visual working memory and feature attention in the model, the working memory content can indirectly exert an effect on perceptual processing in the low-level sensory representation. This in turn biases saccadic movement planning in the spatial pathway, allowing the model to quantitatively reproduce the observed interaction effects. The continuous coupling between representations in the model also implies that modulation should be bidirectional, and model simulations provide specific predictions for complementary effects of saccade target selection on visual working memory. These predictions were empirically confirmed in a new experiment: Memory for a sample color was biased toward the color of a task-irrelevant saccade target object, demonstrating the bidirectional coupling between visual working memory and perceptual processing. PMID:25228628

  9. Analysis of mucolipidosis II/III GNPTAB missense mutations identifies domains of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase involved in catalytic function and lysosomal enzyme recognition.

    PubMed

    Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather; Yox, Alex; Steet, Richard; Kornfeld, Stuart

    2015-01-30

    UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits. PMID:25505245

  10. X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal-lysosomal dysfunction.

    PubMed

    Strmme, Petter; Dobrenis, Kostantin; Sillitoe, Roy V; Gulinello, Maria; Ali, Nafeeza F; Davidson, Cristin; Micsenyi, Matthew C; Stephney, Gloria; Ellevog, Linda; Klungland, Arne; Walkley, Steven U

    2011-11-01

    Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium-hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium-hydrogen exchanger 6 deficiency would most likely disrupt the endosomal-lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium-hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for ?-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium-hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal-lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert could provide a unified explanation for the cellular and clinical phenotypes in humans with SLC9A6 mutations. PMID:21964919

  11. X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal–lysosomal dysfunction

    PubMed Central

    Strømme, Petter; Dobrenis, Kostantin; Sillitoe, Roy V.; Gulinello, Maria; Ali, Nafeeza F.; Davidson, Cristin; Micsenyi, Matthew C.; Stephney, Gloria; Ellevog, Linda; Klungland, Arne

    2011-01-01

    Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium–hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium–hydrogen exchanger 6 deficiency would most likely disrupt the endosomal–lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium–hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for β-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium–hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal–lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert could provide a unified explanation for the cellular and clinical phenotypes in humans with SLC9A6 mutations. PMID:21964919

  12. THINK OUTSIDE THE COLOR BOX: PROBABILISTIC TARGET SELECTION AND THE SDSS-XDQSOQUASAR TARGETING CATALOG

    SciTech Connect

    BOVY, J.; Sheldon, E.; Hennawi, J.F.; Hogg, D.W.; Myers, A.D.; et al.

    2011-03-10

    We present the SDSS-XDQSO quasar targeting catalog for efficient flux-based quasar target selection down to the faint limit of the Sloan Digital Sky Survey (SDSS) catalog, even at medium redshifts (2.5 {approx}< z {approx}< 3) where the stellar contamination is significant. We build models of the distributions of stars and quasars in flux space down to the flux limit by applying the extreme-deconvolution method to estimate the underlying density. We convolve this density with the flux uncertainties when evaluating the probability that an object is a quasar. This approach results in a targeting algorithm that is more principled, more efficient, and faster than other similar methods. We apply the algorithm to derive low-redshift (z < 2.2), medium-redshift (2.2 {le} z {le} 3.5), and high-redshift (z > 3.5) quasar probabilities for all 160,904,060 point sources with dereddened i-band magnitude between 17.75 and 22.45 mag in the 14,555 deg{sup 2} of imaging from SDSS Data Release 8. The catalog can be used to define a uniformly selected and efficient low- or medium-redshift quasar survey, such as that needed for the SDSS-III's Baryon Oscillation Spectroscopic Survey project. We show that the XDQSO technique performs as well as the current best photometric quasar-selection technique at low redshift, and outperforms all other flux-based methods for selecting the medium-redshift quasars of our primary interest. We make code to reproduce the XDQSO quasar target selection publicly available.

  13. SELECTION, PRIORITIZATION, AND CHARACTERISTICS OF KEPLER TARGET STARS

    SciTech Connect

    Batalha, Natalie M.; Borucki, William J.; Koch, David G.; Bryson, Stephen T.; Haas, Michael R.; Brown, Timothy M.; Caldwell, Douglas A.; Hall, Jennifer R.; Latham, David W.; Meibom, Soren; Monet, David G.

    2010-04-20

    The Kepler Mission began its 3.5 year photometric monitoring campaign in 2009 May on a select group of approximately 150,000 stars. The stars were chosen from the {approx} half million in the field of view that are brighter than 16th magnitude. The selection criteria are quantitative metrics designed to optimize the scientific yield of the mission with regard to the detection of Earth-size planets in the habitable zone. This yields more than 90,000 G-type stars on or close to the main sequence, >20, 000 of which are brighter than 14th magnitude. At the temperature extremes, the sample includes approximately 3000 M-type dwarfs and a small sample of O- and B-type MS stars (<200). The small numbers of giants are included in the sample: {approx}5000 stars with surface gravities log(g) < 3.5. We present a brief summary of the selection process and the stellar populations it yields in terms of surface gravity, effective temperature, and apparent magnitude. In addition to the primary, statistically derived target set, several ancillary target lists were manually generated to enhance the science of the mission, examples being: known eclipsing binaries, open cluster members, and high proper motion stars.

  14. Engineering novel cell surface chemistry for selective tumor cell targeting

    SciTech Connect

    Bertozzi, C.R.

    1997-12-31

    A common feature of many different cancers is the high expression level of the two monosaccharides sialic acid and fucose within the context of cell-surface associated glycoconjugates. A correlation has been made between hypersialylation and/or hyperfucosylation and the highly metastatic phenotype. Thus, a targeting strategy based on sialic acid or fucose expression would be a powerful tool for the development of new cancer cell-selective therapies and diagnostic agents. We have discovered that ketone groups can be incorporated metabolically into cell-surface associated sialic acids. The ketone is can be covalently ligated with hydrazide functionalized proteins or small molecules under physiological conditions. Thus, we have discovered a mechanism to selectively target hydrazide conjugates to highly sialylated cells such as cancer cells. Applications of this technology to the generation of novel cancer cell-selective toxins and MRI contrast reagents will be discussed, in addition to progress towards the use of cell surface fucose residues as vehicles for ketone expression.

  15. Selective targeting of the stress chaperome as a therapeutic strategy

    PubMed Central

    Taldone, Tony; Ochiana, Stefan O.; Patel, Pallav D.; Chiosis, Gabriela

    2014-01-01

    Normal cellular function is maintained by coordinated proteome machinery that performs a vast array of activities. Helping the proteome in such roles is the chaperome, a network of molecular chaperones and folding enzymes. The stressed cell contains, at any time, a complex mixture of chaperome complexes; a majority performs housekeeping functions similarly to non-stressed, normal cells, but a finely-tuned fraction buffers the proteome altered by chronic stress. The stress chaperome is epigenetically distinct from its normal, housekeeping counterpart, providing a basis for its selective targeting by small molecules. Here we discuss development of chaperome inhibitors, and how agents targeting chaperome members in stressed cells are in fact being directed towards chaperome complexes and their effect is therefore determined by their ability to sample and engage such complexes. A new approach is needed to target and implement chaperome modulators in the investigation of diseases, and we propose that the classical thinking in drug discovery needs adjustment when developing chaperome-targeting drugs. PMID:25262919

  16. Target coverage and selectivity in field steering brain stimulation.

    PubMed

    Cubo, Ruben; strom, Mattias; Medvedev, Alexander

    2014-01-01

    Deep Brain Stimulation (DBS) is an established treatment in Parkinson's Disease. The target area is defined based on the state and brain anatomy of the patient. The stimulation delivered via state-of-the-art DBS leads that are currently in clinical use is difficult to individualize to the patient particularities. Furthermore, the electric field generated by such a lead has a limited selectivity, resulting in stimulation of areas adjacent to the target and thus causing undesirable side effects. The goal of this study is, using actual clinical data, to compare in silico the stimulation performance of a symmetrical generic lead to a more versatile and adaptable one allowing, in particular, for asymmetric stimulation. The fraction of the volume of activated tissue in the target area and the fraction of the stimulation field that spreads beyond it are computed for a clinical data set of patients in order to quantify the lead performance. The obtained results suggest that using more versatile DBS leads might reduce the stimulation area beyond the target and thus lessen side effects for the same achieved therapeutical effect. PMID:25570011

  17. Isolation and characterization of chicken liver lysosomes.

    PubMed

    Nakabayashi, T; Ikezawa, H

    1988-06-01

    The distribution patterns of chicken liver lysosomal enzymes were studied in iso-osmotic gradients of Percoll. The lysosomal enzymes separated by Percoll gradients showed three different types of distribution. In contrast with rat liver lysosomes, purified chicken liver lysosomes were very stable during storage at 4 degrees C. PMID:2845991

  18. Selective follicular targeting by modification of the particle sizes.

    PubMed

    Patzelt, Alexa; Richter, Heike; Knorr, Fanny; Schäfer, Ulrich; Lehr, Claus-Michael; Dähne, Lars; Sterry, Wolfram; Lademann, Juergen

    2011-02-28

    Hair follicles represent interesting target sites for topically applied substances such as topical vaccinations or agents used in the field of regenerative medicine. In recent years, it could be shown that particles penetrate very effectively into the hair follicles. In the present study, the influence of particle size on the follicular penetration depths was examined. The penetration depths of two different types of particles sized 122 to 1000 nm were determined in vitro on porcine skin. The results revealed that the particles of medium size (643 and 646 nm, respectively) penetrated deeper into the porcine hair follicles than smaller or larger particles. It was concluded that by varying the particle size, different sites within the porcine hair follicle can be targeted selectively. For the human terminal hair follicle, the situation can be expected to be similar due to a similar size ratio of the hair follicles. PMID:21087645

  19. Wavelength band selection method for multispectral target detection.

    PubMed

    Karlholm, Jrgen; Renhorn, Ingmar

    2002-11-10

    A framework is proposed for the selection of wavelength bands for multispectral sensors by use of hyperspectral reference data. Using the results from the detection theory we derive a cost function that is minimized by a set of spectral bands optimal in terms of detection performance for discrimination between a class of small rare targets and clutter with known spectral distribution. The method may be used, e.g., in the design of multispectral infrared search and track and electro-optical missile warning sensors, where a low false-alarm rate and a high-detection probability for detection of small targets against a clutter background are of critical importance, but the required high frame rate prevents the use of hyperspectral sensors. PMID:12440532

  20. A negative selection heuristic to predict new transcriptional targets

    PubMed Central

    2013-01-01

    Background Supervised machine learning approaches have been recently adopted in the inference of transcriptional targets from high throughput trascriptomic and proteomic data showing major improvements from with respect to the state of the art of reverse gene regulatory network methods. Beside traditional unsupervised techniques, a supervised classifier learns, from known examples, a function that is able to recognize new relationships for new data. In the context of gene regulatory inference a supervised classifier is coerced to learn from positive and unlabeled examples, as the counter negative examples are unavailable or hard to collect. Such a condition could limit the performance of the classifier especially when the amount of training examples is low. Results In this paper we improve the supervised identification of transcriptional targets by selecting reliable counter negative examples from the unlabeled set. We introduce an heuristic based on the known topology of transcriptional networks that in fact restores the conventional positive/negative training condition and shows a significant improvement of the classification performance. We empirically evaluate the proposed heuristic with the experimental datasets of Escherichia coli and show an example of application in the prediction of BCL6 direct core targets in normal germinal center human B cells obtaining a precision of 60%. Conclusions The availability of only positive examples in learning transcriptional relationships negatively affects the performance of supervised classifiers. We show that the selection of reliable negative examples, a practice adopted in text mining approaches, improves the performance of such classifiers opening new perspectives in the identification of new transcriptional targets. PMID:23368951

  1. Neuropathic Lysosomal Storage Disorders

    PubMed Central

    Pastores, Gregory M.; Maegawa, Gustavo H.B.

    2014-01-01

    The lysosomal storage disorders (LSDs) are a clinically heterogeneous group of inborn errors of metabolism, associated with the accumulation of incompletely degraded macromolecules within several cellular sites. Affected individuals present with a broad range of clinical problems, including hepatosplenomegaly and skeletal dysplasia. Onset of symptoms may range from birth to adulthood. The majority are associated with neurological features, including developmental delay, behavioral/psychiatric disturbances, seizures, acroparesthesia, motor weakness, cerebrovascular ischemic events and extra-pyramidal signs. It should be noted that later-onset forms are often misdiagnosed as symptoms, which might include psychiatric manifestations, are slowly progressive and may precede other neurologic or systemic features. Inheritance is primarily autosomal recessive. For all subtypes, diagnosis can be confirmed using a combination of biochemical and/or molecular assays. In a few LSDs, treatment with either hematopoietic stem cell transplantation, enzyme replacement or substrate reduction therapy is available. Genetic counseling is important, so patients and their families can be informed of reproductive risks, disease prognosis and therapeutic options. Investigations of disease mechanisms are providing insights into potential therapeutic approaches. Symptomatic care, which remains the mainstay for most subtypes, can lead to significant improvement in quality of life. PMID:24176423

  2. Sphingolipids and lysosomal pathologies.

    PubMed

    Schulze, Heike; Sandhoff, Konrad

    2014-05-01

    Endocytosed (glyco)sphingolipids are degraded, together with other membrane lipids in a stepwise fashion by endolysosomal enzymes with the help of small lipid binding proteins, the sphingolipid activator proteins (SAPs), at the surface of intraluminal lysosomal vesicles. Inherited defects in a sphingolipid-degrading enzyme or SAP cause the accumulation of the corresponding lipid substrates, including cytotoxic lysosphingolipids, such as galactosylsphingosine and glucosylsphingosine, and lead to a sphingolipidosis. Analysis of patients with prosaposin deficiency revealed the accumulation of intra-endolysosmal vesicles and membrane structures (IM). Feeding of prosaposin reverses the storage, suggesting inner membrane structures as platforms of sphingolipid degradation. Water soluble enzymes can hardly attack sphingolipids embedded in the membrane of inner endolysosomal vesicles. The degradation of sphingolipids with few sugar residues therefore requires the help of the SAPs, and is strongly stimulated by anionic membrane lipids. IMs are rich in anionic bis(monoacylglycero)phosphate (BMP). This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology. PMID:24184515

  3. Target Search and Selection for the DI/EPOXI Spacecraft

    NASA Technical Reports Server (NTRS)

    Grebow, Daniel J.; Bhaskaran, Shyam; Chesley, Steven R.

    2012-01-01

    Upon completion of the Hartley 2 flyby in November 2010, the Deep Impact (DI) spacecraft resided in a solar orbit without possibility for gravity assist with any large body. Conservative estimates of remaining fuel were enough to provide only an 18 m/s impulse on the spacecraft. We present our method and results of our systematic scan of potential small body encounters for DI, and our criteria to narrow the selection to the asteroid 2002 GT as the target flyby body. The mission profile has two deterministic maneuvers to achieve the encounter, the first of which executed on November 25, 2011.

  4. Target Search & Selection for the DI/EPOXI Spacecraft

    NASA Technical Reports Server (NTRS)

    Grebow, Daniel J.; Bhaskaran, Shyam; Chesley, Steven R.

    2012-01-01

    Upon completion of the Hartley 2 flyby in November 2010, the Deep Impact (DI) spacecraft resided in a solar orbit without possibility for gravity assist with any large body. Conservative estimates of remaining fuel were enough to provide only an 18 m/s impulse on the spacecraft. We present our method and results of our systematic scan of potential small body encounters for DI, and our criteria to narrow the selection to the asteroid 2002 GT as the target flyby body. The mission profile has two deterministic maneuvers to achieve the encounter, the first of which executed on November 25, 2011.

  5. Target selective tilt-after effect during texture learning.

    PubMed

    Harris, Hila; Pinchuk-Yacobi, Noga; Sagi, Dov

    2015-09-01

    In texture learning, observers are presented with repeated stimulations, resulting in within-day threshold elevation, as well as between day threshold reductions. Within-day deteriorations were shown to be location and orientation specific (Mednick et. al., 2002; Ofen et al., 2007). Accordingly, the declined performance was suggested to reflect sensory adaptation. Here we test whether extensive training produces adaptation dependent tilt after-effects (TAE). Six observers were trained for 5 days on the texture discrimination task (Karni & Sagi, 1991), 800-1000 trials/day. The target (40ms) was composed of 3 lines of 22.5o orientation, stacked vertically or horizontally (2AFC task), embedded in a background of -22.5o lines. The target was followed by a variable blank interval and a mask (100ms). Multiple tests of perceived vertical (PV) were carried out prior and after each daily session to evaluate TAE at four locations, corresponding to: a target line, +22.5o on all trials (T+); both target and background lines, balanced, either +22.5o or -22.5o (T0); and the two background locations, near (BN) and far from the target (BF). Results showed learning across days, with within-day deterioration that varied across days. There was a significant TAE immediately following TDT training at both target's locations, at T+ (-1.60.3, meanSEM; p< 0.01) and, more surprisingly, at T0 (-0.80.2; p=0.03) but not at background locations (BN, 0.30.3; BF, 0.30.1). The persistence of the TAE varied across days, as indicated by successive PV tests; decaying faster at location T+ (p=0.02), while persisting longer at location T0 (p=0.02). Here we show that texture training induces a localized target-selective TAE, which in return has a training-dependent component. The absence of background TAE and the target-biased TAE at the balanced location indicate that aftereffects are not determined by stimulus statistics, but rather by experience-dependent task-relevance. This supports mutual interactions between sensory adaptation and perceptual learning. Meeting abstract presented at VSS 2015. PMID:26326822

  6. Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents.

    PubMed

    Brojatsch, Jürgen; Lima, Heriberto; Palliser, Deborah; Jacobson, Lee S; Muehlbauer, Stefan M; Furtado, Raquel; Goldman, David L; Lisanti, Michael P; Chandran, Kartik

    2015-01-01

    Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis. PMID:25830414

  7. Lysosomal dysfunction and impaired autophagy in a novel mouse model deficient for the lysosomal membrane protein Cln7.

    PubMed

    Brandenstein, Laura; Schweizer, Michaela; Sedlacik, Jan; Fiehler, Jens; Storch, Stephan

    2016-02-15

    CLN7 disease is an autosomal recessive, childhood-onset neurodegenerative lysosomal storage disorder caused by the defective lysosomal membrane protein CLN7. We have disrupted the Cln7/Mfsd8 gene in mice by targeted deletion of exon 2 generating a novel knockout (KO) mouse model for CLN7 disease, which recapitulates key features of human CLN7 disease pathology. Cln7 KO mice showed increased mortality and a neurological phenotype including hind limb clasping and myoclonus. Lysosomal dysfunction in the brain of mutant mice was shown by the storage of autofluorescent lipofuscin-like lipopigments, subunit c of mitochondrial ATP synthase and saposin D and increased expression of lysosomal cathepsins B, D and Z. By immunohistochemical co-stainings, increased cathepsin Z expression restricted to Cln7-deficient microglia and neurons was found. Ultrastructural analyses revealed large storage bodies in Purkinje cells of Cln7 KO mice containing inclusions composed of irregular, curvilinear and rectilinear profiles as well as fingerprint profiles. Generalized astrogliosis and microgliosis in the brain preceded neurodegeneration in the olfactory bulb, cerebral cortex and cerebellum in Cln7 KO mice. Increased levels of LC3-II and the presence of neuronal p62- and ubiquitin-positive protein aggregates suggested that impaired autophagy represents a major pathomechanism in the brain of Cln7 KO mice. The data suggest that loss of the putative lysosomal transporter Cln7 in the brain leads to lysosomal dysfunction, impaired constitutive autophagy and neurodegeneration late in disease. PMID:26681805

  8. Selectively Targeting Prostate Cancer with Antiandrogen Equipped Histone Deacetylase Inhibitors

    PubMed Central

    Gryder, Berkley E.; Akbashev, Michelle J.; Rood, Michael K.; Raftery, Eric D.; Meyers, Warren M.; Dillard, Paulette; Khan, Shafiq; Oyelere, Adegboyega K.

    2013-01-01

    Diverse cellular processes relevant to cancer progression are regulated by the acetylation status of proteins. Among such processes is chromatin remodeling via histone proteins, controlled by opposing histone deacetylase (HDAC) and histone acetyltransferase (HAT) enzymes. Histone deacetylase inhibitors (HDACi) show great promise in preclinical cancer models, but clinical trials treating solid tumors have failed to improve patient survival. This is due in part to an inability of HDACi to effectively accumulate in cancerous cells. To address this problem we designed HDACi with secondary pharmacophores to facilitate selective accumulation in malignant cells. We present the first example of HDACi compounds targeted to prostate tumors by equipping them with the additional ability to bind the androgen receptor (AR) with non-steroidal antiandrogen moieties. Leads among these new dual-acting molecules bind to the AR and halt AR transcriptional activity at lower concentrations than clinical antiandrogens. They inhibit key isoforms of HDAC with low nanomolar potency. Fluorescent microscopy reveals varying degrees of AR nuclear localization in response to these compounds that correlates with their HDAC activity. These biological properties translate into potent anticancer activity against hormone dependent (AR+) LNCaP and to a lesser extent against hormone independent (AR−) DU145 prostate cancer, while having greatly reduced toxicity in non-cancerous cells. This illustrates that engaging multiple biological targets with a single chemical probe can achieve both potent and cell-type selective responses. PMID:24004176

  9. Positive–negative-selection-mediated gene targeting in rice

    PubMed Central

    Shimatani, Zenpei; Nishizawa-Yokoi, Ayako; Endo, Masaki; Toki, Seiichi; Terada, Rie

    2015-01-01

    Gene targeting (GT) refers to the designed modification of genomic sequence(s) through homologous recombination (HR). GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ) occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive–negative selection (PNS) is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice—a major crop worldwide—reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology based on PNS are discussed. PMID:25601872

  10. Integration target site selection by a resurrected human endogenous retrovirus

    PubMed Central

    Brady, Troy; Lee, Young Nam; Ronen, Keshet; Malani, Nirav; Berry, Charles C.; Bieniasz, Paul D.; Bushman, Frederic D.

    2009-01-01

    At least 8% of the human genome was formed by integration of retroviral DNA sequences. Here we analyze the forces directing the accumulation of human endogenous retroviruses (HERVs) by comparing de novo HERV integration targeting with the distribution of fixed HERV elements in the human genome. All known genomic HERVs are inactive due to mutation, but we were able to study integration targeting using a reconstituted consensus HERV-K (designated HERV-KCon). We found that HERV-KCon integrated preferentially in transcription units, in gene-rich regions, and near features associated with active transcription units and associated regulatory regions. In contrast, genomic HERV-K proviruses are found preferentially outside transcription units. The minority of genomic HERVKs present inside transcription units are in opposite transcriptional orientation relative to the host gene, the orientation predicted to be minimally disruptive to host mRNA synthesis, but de novo HERV-KCon integration within transcription units showed no orientation bias. We also found that the youngest HERV-K elements in the human genome showed a distribution intermediate between de novo HERV-KCon integration sites and older fixed HERV-Ks. These findings indicate that accumulation of HERVs in the human germline is a two-step process: integration targeting biases direct initial accumulation, then purifying selection leads to loss of proviruses disrupting gene function. PMID:19270161

  11. Landslide susceptibility mapping in three selected target zones in Afghanistan

    NASA Astrophysics Data System (ADS)

    Schwanghart, Wolfgang; Seegers, Joe; Zeilinger, Gerold

    2015-04-01

    In May 2014, a large and mobile landslide destroyed the village Ab Barek, a village in Badakshan Province, Afghanistan. The landslide caused several hundred fatalities and once again demonstrated the vulnerability of Afghanistan's population to extreme natural events following more than 30 years of civil war and violent conflict. Increasing the capacity of Afghanistan's population by strengthening the disaster preparedness and management of responsible government authorities and institutions is thus a major component of international cooperation and development strategies. Afghanistan is characterized by high relief and widely varying rock types that largely determine the spatial distribution as well as emplacement modes of mass movements. The major aim of our study is to characterize this variability by conducting a landslide susceptibility analysis in three selected target zones: Greater Kabul Area, Badakhshan Province and Takhar Province. We expand on an existing landslide database by mapping landforms diagnostic for landslides (e.g. head scarps, normal faults and tension cracks), and historical landslide scars and landslide deposits by visual interpretation of high-resolution satellite imagery. We conduct magnitude frequency analysis within subregional physiogeographic classes based on geological maps, climatological and topographic data to identify regional parameters influencing landslide magnitude and frequency. In addition, we prepare a landslide susceptibility map for each area using the Weight-of-Evidence model. Preliminary results show that the three selected target zones vastly differ in modes of landsliding. Low magnitude but frequent rockfall events are a major hazard in the Greater Kabul Area threatening buildings and infrastructure encroaching steep terrain in the city's outskirts. Mass movements in loess covered areas of Badakshan are characterized by medium to large magnitudes. This spatial variability of characteristic landslide magnitudes and modes of emplacement necessitates different strategies to assess, mitigate, and prepare for landslides in the three different target zones.

  12. Signals from the lysosome: a control centre for cellular clearance and energy metabolism.

    PubMed

    Settembre, Carmine; Fraldi, Alessandro; Medina, Diego L; Ballabio, Andrea

    2013-05-01

    For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease. PMID:23609508

  13. Mannose 6 phosphorylation of lysosomal enzymes controls B cell functions

    PubMed Central

    Schweizer, Michaela; Kollmann, Katrin; Schumacher, Vala; Muschol, Nicole; Tolosa, Eva; Mittrcker, Hans-Willi

    2015-01-01

    Antigen processing and presentation and cytotoxic targeting depend on the activities of several lysosomal enzymes that require mannose 6-phosphate (M6P) sorting signals for efficient intracellular transport and localization. In this paper, we show that mice deficient in the formation of M6P residues exhibit significant loss of cathepsin proteases in B cells, leading to lysosomal dysfunction with accumulation of storage material, impaired antigen processing and presentation, and subsequent defects in B cell maturation and antibody production. The targeting of lysosomal and granular enzymes lacking M6P residues is less affected in dendritic cells and T cells and sufficient for maintenance of degradative and lytic functions. M6P deficiency also impairs serum immunoglobulin levels and antibody responses to vaccination in patients. Our data demonstrate the critical role of M6P-dependent transport routes for B cell functions in vivo and humoral immunity in mice and human. PMID:25601403

  14. Mitochondrial genomes are retained by selective constraints on protein targeting.

    PubMed

    Bjrkholm, Patrik; Harish, Ajith; Hagstrm, Erik; Ernst, Andreas M; Andersson, Siv G E

    2015-08-18

    Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome. PMID:26195779

  15. Mitochondrial genomes are retained by selective constraints on protein targeting

    PubMed Central

    Björkholm, Patrik; Harish, Ajith; Hagström, Erik; Ernst, Andreas M.; Andersson, Siv G. E.

    2015-01-01

    Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome. PMID:26195779

  16. Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase.

    PubMed Central

    Tikkanen, R; Peltola, M; Oinonen, C; Rouvinen, J; Peltonen, L

    1997-01-01

    Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase. PMID:9362483

  17. Cancer Immunotherapy: Selected Targets and Small-Molecule Modulators.

    PubMed

    Weinmann, Hilmar

    2016-03-01

    There is a significant amount of excitement in the scientific community around cancer immunotherapy, as this approach has renewed hope for many cancer patients owing to some recent successes in the clinic. Currently available immuno-oncology therapeutics under clinical development and on the market are mostly biologics (antibodies, proteins, engineered cells, and oncolytic viruses). However, modulation of the immune system with small molecules offers several advantages that may be complementary and potentially synergistic to the use of large biologicals. Therefore, the discovery and development of novel small-molecule modulators is a rapidly growing research area for medicinal chemists working in cancer immunotherapy. This review provides a brief introduction into recent trends related to selected targets and pathways for cancer immunotherapy and their small-molecule pharmacological modulators. PMID:26836578

  18. Selective Targeting of TGF-? Activation to Treat Fibroinflammatory Airway Disease

    PubMed Central

    Minagawa, Shunsuke; Lou, Jianlong; Seed, Robert I.; Cormier, Anthony; Wu, Shenping; Cheng, Yifan; Murray, Lynne; Tsui, Ping; Connor, Jane; Herbst, Ronald; Govaerts, Cedric; Barker, Tyren; Cambier, Stephanie; Yanagisawa, Haruhiko; Goodsell, Amanda; Hashimoto, Mitsuo; Brand, Oliver J.; Cheng, Ran; Ma, Royce; McKnelly, Kate J.; Wen, Weihua; Hill, Arthur; Jablons, David; Wolters, Paul; Kitamura, Hideya; Araya, Jun; Barczak, Andrea J.; Erle, David J.; Reichardt, Louis F.; Marks, James D.; Baron, Jody L.; Nishimura, Stephen L.

    2015-01-01

    Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor? (TGF-?) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-? is expressed in a latent form that requires activation. The integrin ?v?8 (encoded by the itgb8 gene) is a receptor for latent TGF-? and is essential for its activation. Expression of integrin ?v?8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human ?v?8 (B5) inhibited TGF-? activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-? activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that ?v?8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity ?v?8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-? pathway to treat fibroinflammatory airway diseases. PMID:24944194

  19. MESSI: metabolic engineering target selection and best strain identification tool

    PubMed Central

    Kang, Kang; Li, Jun; Lim, Boon Leong; Panagiotou, Gianni

    2015-01-01

    Metabolic engineering and synthetic biology are synergistically related fields for manipulating target pathways and designing microorganisms that can act as chemical factories. Saccharomyces cerevisiaes ideal bioprocessing traits make yeast a very attractive chemical factory for production of fuels, pharmaceuticals, nutraceuticals as well as a wide range of chemicals. However, future attempts of engineering S. cerevisiaes metabolism using synthetic biology need to move towards more integrative models that incorporate the high connectivity of metabolic pathways and regulatory processes and the interactions in genetic elements across those pathways and processes. To contribute in this direction, we have developed Metabolic Engineering target Selection and best Strain Identification tool (MESSI), a web server for predicting efficient chassis and regulatory components for yeast bio-based production. The server provides an integrative platform for users to analyse ready-to-use public high-throughput metabolomic data, which are transformed to metabolic pathway activities for identifying the most efficient S. cerevisiae strain for the production of a compound of interest. As input MESSI accepts metabolite KEGG IDs or pathway names. MESSI outputs a ranked list of S. cerevisiae strains based on aggregation algorithms. Furthermore, through a genome-wide association study of the metabolic pathway activities with the strains natural variation, MESSI prioritizes genes and small variants as potential regulatory points and promising metabolic engineering targets. Users can choose various parameters in the whole process such as (i) weight and expectation of each metabolic pathway activity in the final ranking of the strains, (ii) Weighted AddScore Fuse or Weighted Borda Fuse aggregation algorithm, (iii) type of variants to be included, (iv) variant sets in different biological levels. Database URL: http://sbb.hku.hk/MESSI/ PMID:26255308

  20. Targeting the actin cytoskeleton: selective antitumor action via trapping PKC?.

    PubMed

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-? (PKC?), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKC?, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKC?, we suggest that trapping PKC? via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKC?, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKC? signaling shown in a xenograft breast cancer model. PMID:25165884

  1. Deciphering the Code for Retroviral Integration Target Site Selection

    PubMed Central

    Santoni, Federico Andrea; Hartley, Oliver; Luban, Jeremy

    2010-01-01

    Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses. PMID:21124862

  2. A selective inhibitor Gal-PUGNAc of human lysosomal beta-hexosaminidases modulates levels of the ganglioside GM2 in neuroblastoma cells.

    PubMed

    Stubbs, Keith A; Macauley, Matthew S; Vocadlo, David J

    2009-01-01

    Gal-PUGNAc (see picture), a highly selective inhibitor for beta-hexosaminidases HEXA and HEXB is cell-permeable and modulates the activity of HEXA and HEXB in tissue culture, increasing ganglioside GM2 levels. Gal-PUGNAc should allow the role of these enzymes to be studied at the cellular level without generating a complex chemical phenotype from concomitant inhibition of O-GlcNAcase. PMID:19130519

  3. Uncovering the role of Snapin in regulating autophagy-lysosomal function.

    PubMed

    Cai, Qian; Sheng, Zu-Hang

    2011-04-01

    The autophagy-lysosomal system is the major degradation pathway essential for the maintenance and survival of neurons. This process requires efficient late endocytic transport from distal processes to the soma, in which lysosomes are predominantly localized. However, it is not clear how late endocytic transport has an impact upon neuronal autophagy-lysosomal function. We recently revealed that Snapin acts as a dynein motor adaptor and coordinates retrograde transport and late endosomal-lysosomal trafficking, thus maintaining efficient autophagy-lysosomal function in neurons. Snapin(-/-) neurons display impaired retrograde transport and clustering of late endosomes along neuronal processes, aberrant accumulation of immature lysosomes, and impaired clearance of autolysosomes. Snapin deficiency leads to reduced neuron viability, neurodegeneration, and developmental defects in the central nervous system. Reintroducing the snapin transgene rescues these phenotypes by enhancing the delivery of endosomal cargos to lysosomes and by facilitating autophagy-lysosomal function. Our study suggests that Snapin is a candidate molecular target for autophagy-lysosomal regulation. PMID:21233602

  4. Lysosomal abnormalities during benzo(a)pyrene-induced experimental lung carcinogenesis--defensive role of capsaicin.

    PubMed

    Anandakumar, P; Kamaraj, S; Jagan, S; Ramakrishnan, G; Devaki, T

    2009-02-01

    The objective of the present study was to investigate whether lysosome is a target in benzo(a)pyrene-induced, oxidative stress-mediated lung cancer in Swiss albino mice and the plausible role of the phytochemical substance capsaicin in mitigating lysosomal damage. Oxidative stress was assessed based on the level of carbonyl content. The activities of lysosomal proteases like cathepsin-D, cathepsin-B, beta-D-glucosidase, beta-D-galactosidase, beta-D-glucuronidase, beta-D-N-acetylglucosaminidase and acid phosphatase were assessed to evaluate lysosomal function. Administration of benzo(a)pyrene (50 mg/kg body weight) to mice induced a increase in the activities of lysosomal enzymes and oxidative stress was evident by the increase in carbonyl content. Treatment with capsaicin (10 mg/kg body weight) decreased carbonyl content and restored the activities of lysosomal enzymes to near normalcy. Transmission electron microscopic study of lysosomes further showed the defensive action of capsaicin against the lysosomal damage caused in benzo(a)pyrene-induced lung cancer. From the present study, it can be concluded that lysosomal damage is an indispensable event in benzo(a)pyrene-induced lung cancer, and capsaicin was able to effectively prevent it, which proves the chemoprotective effect of capsaicin against benzo(a)pyrene-induced experimental lung carcinogenesis. PMID:19207542

  5. Enhancing the efficacy of hormonal agents with selected targeted agents.

    PubMed

    Johnston, Stephen R D

    2009-06-01

    Several selected targeted agents are being investigated in combination with endocrine therapy for patients with breast cancer in an attempt to overcome or prevent endocrine resistance. The role of type I growth factor receptors epidermal growth factor receptor (EGFR) and HER2 in cross-talk with estrogen receptor (ER) signaling has been confirmed in preclinical studies in which various inhibitors have yielded additive or synergistic effects when combined with endocrine agents. Recently, several results from clinical trials investigating this concept have been reported. In ER-positive/HER-positive advanced breast cancer, the addition of trastuzumab to the aromatase inhibitor anastrozole, or the tyrosine kinase inhibitor (TKI) lapatinib to letrozole, both have significantly improved progression-free survival (PFS). The EGFR TKI gefitinib combined with tamoxifen as first-line therapy for ER-positive metastatic disease improved PFS (but not objective response rate) for patients with no previous endocrine therapy or completion of previous adjuvant therapy. A second study in a similar setting showed significant improvement in PFS for gefitinib plus anastrozole. Although it is encouraging that this approach could delay resistance, only a small proportion of patients benefit. Attempts to identify likely responders have been made in the neoadjuvant setting, with pre- and post-treatment biopsies being used to study biomarker changes. A recent preoperative study of letrozole with or without the mammalian target of rapamycin (mTOR) inhibitor everolimus reported greater tumor shrinkage for the combination, with changes in proliferation being predictive for response together with strong expression of protein S6 kinase, a downstream marker of activated mTOR. Key aspects that need to be addressed in future trials include understanding the mechanisms of action for each novel agent, designing the best trial and endpoints to demonstrate added benefit, and ensuring appropriately stratified populations based on previous endocrine exposure and/or sensitivity. PMID:19561004

  6. DNA Aptamers Selectively Target Leishmania infantum H2A Protein

    PubMed Central

    Martn, M. Elena; Garca-Hernndez, Marta; Garca-Recio, Eva M.; Gmez-Chacn, Gernimo F.; Snchez-Lpez, Marta; Gonzlez, Vctor M.

    2013-01-01

    Parasites of the genus Leishmania produce leishmaniasis which affects millions people around the world. Understanding the molecular characteristics of the parasite can increase the knowledge about the mechanisms underlying disease development and progression. Thus, the study of the molecular features of histones has been considered of particular interest because Leishmania does not condense the chromatin during mitosis and, consequently, a different role for these proteins in the biology of the parasite can be expected. Furthermore, the sequence divergences in the amino and in the carboxy-terminal domains of the kinetoplastid core histones convert them in potential diagnostic and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania histones is essential for the progress of this kind of study. Two aptamers which specifically recognize Leishmania infantum H2A histone were cloned from a previously obtained ssDNA enriched population. These aptamers were sequenced and subjected to an in silico analysis. ELONA, slot blot and Western blot were performed to establish aptamer affinity and specificity for LiH2A histone and ELONA assays using peptides corresponding to overlapped sequences of LiH2A were made mapping the aptamers:LiH2A interaction. As proofs of concept, aptamers were used to determine the number of parasites in an ELONA platform and to purify LiH2A from complex mixtures. The aptamers showed different secondary structures among them; however, both of them were able to recognize the same peptides located in a side of the protein. In addition, we demonstrate that these aptamers are useful for LiH2A identification and also may be of potential application as diagnostic system and as a laboratory tool with purification purpose. PMID:24205340

  7. Cathepsin Inhibition-Induced Lysosomal Dysfunction Enhances Pancreatic Beta-Cell Apoptosis in High Glucose

    PubMed Central

    Jung, Minjeong; Lee, Jaemeun; Seo, Hye-Young; Lim, Ji Sun; Kim, Eun-Kyoung

    2015-01-01

    Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic ?-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic ?-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of ?-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic ?-cells. PMID:25625842

  8. The Nutrient-Responsive Transcription Factor TFE3, Promotes Autophagy, Lysosomal Biogenesis, and Clearance of Cellular Debris

    PubMed Central

    Martina, José A.; Diab, Heba I.; Lishu, Li; Jeong-A, Lim; Patange, Simona; Raben, Nina; Puertollano, Rosa

    2015-01-01

    The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator TFEB revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. Here, we report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag GTPases and exhibited mTORC1-dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. Following starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders. PMID:24448649

  9. Signaling from lysosomes to mitochondria sensitizes cancer cells to photodynamic treatment

    NASA Astrophysics Data System (ADS)

    Hung, Hsin-I.; Quiogue, Geraldine; Lemasters, John J.; Nieminen, Anna-Liisa

    2011-02-01

    Previously, we showed that photosensitizers that localize to lysosomes are more effective in killing cancer cells than ones directed to mitochondria after photodynamic treatment (PDT). The photosensitizer, phthalocyanine 4 (Pc 4), localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. However, analogues of Pc 4 (e.g., Pc 181) that primarily target lysosomes still produce mitochondria-mediated cell death, although the time course is slower compared to Pc 4-PDT. In A431 epidermoid carcinoma cells, these new analogues preferentially localized in lysosomes were highly efficient in inducing apoptotic cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself was not toxic but greatly enhanced Pc 4-PDT-induced mitochondrial depolarization and cell killing. Both depolarization and cell killing were substantially prevented by iron chelators. The fact that Pc 4-PDT plus bafilomycin treatment did not induce lysosomal membrane damage prior to mitochondrial depolarization suggests that bafilomycin instead induced release of redox active iron from lysosomes into the cytosol that further translocated into mitochondria, where iron-mediated free radical formation occurred. In conclusion, agents that disturb lysosomal function could potentially be used as adjuvants with mitochondrion-targeted photosensitizers to enhance phototoxicity.

  10. Lysosomes relax in the cellular suburbs.

    PubMed

    Gowrishankar, Swetha; Ferguson, Shawn M

    2016-03-14

    Lysosomes support cellular homeostasis by degrading macromolecules and recycling nutrients. In this issue, Johnson et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507112) reveal a heterogeneity in lysosomal pH and degradative ability that correlates with lysosome subcellular localization, raising questions about the functional implications and mechanisms underlying these observations. PMID:26975848

  11. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  12. Selected attributes of polyphenols in targeting oxidative stress in cancer.

    PubMed

    Stepanic, Visnja; Gasparovic, Ana Cipak; Troselj, Koraljka Gall; Amic, Dragan; Zarkovic, Neven

    2015-01-01

    Various plant polyphenols have been recognized as redox active molecules. This review discusses some aspects of polyphenols' modes of redox action, corresponding structure-activity relationships and their potential to be applied as adjuvants to conventional cytostatic drugs. Polyphenols' antioxidative capacity has been discussed as the basis for targeting oxidative stress and, consequently, for their chemopreventive and anti-inflammatory activities, which may alleviate side-effects on normal cells arising from oxidative stress caused by cytostatics. Some polyphenols may scavenge various free radicals directly, and some of them are found to suppress free radical production through inhibiting NADPH oxidases and xanthine oxidase. Additionally, polyphenols may increase antioxidative defense in normal cells by increasing the activity of NRF2, transcription factor for many protective proteins. The activation of the NRF2-mediated signaling pathways in cancer cells results in chemoresistance. Luteolin, apigenin and chrysin reduce NRF2 expression and increase the chemosensitivity of cancer cells to cytostatic drugs. Their common 5,7-dihydroxy-4H-chromen-4-one moiety, may represent a starting pharmacophore model for designing novel, non-toxic compounds for overcoming chemoresistance. However, prooxidative activity of some polyphenols (quercetin, EGCG) may also provide a basis for their use as chemotherapeutic adjuvants since they may enhance cytotoxic effects of cytostatics selectively on cancer cells. However, considerable caution is needed in applying polyphenols to anticancer therapy, since their effects greatly depend on the applied dose, the cell type, exposure time and environmental conditions. PMID:25665579

  13. Human hair follicle: reservoir function and selective targeting.

    PubMed

    Blume-Peytavi, U; Vogt, A

    2011-10-01

    Penetration of topically applied compounds may occur via the stratum corneum, skin appendages and hair follicles. The follicular infundibulum increases the surface area, disrupts the epidermal barrier towards the lower parts of the follicle, and serves as a reservoir. Topical delivery of active compounds to specific targets within the skin, especially to distinct hair follicle compartments or cell populations, may help to treat local inflammatory reactions selectively, with reduced systemic side-effects. Various in vitro and in vivo methods exist for studying the hair follicle structure and follicular penetration pathways. These include cyanoacrylate skin surface stripping, confocal microscopy and cyanoacrylate scalp follicle biopsy. The complex anatomical structure as well as the cyclical activity of the hair follicle must be taken into consideration when designing delivery systems. In addition, delivery into and retention inside the infundibular reservoir are controlled by, for example, molecule or particle size, their polarity and the type of preparation. Preferred penetration depth and storage time must also be considered. Particles with release mechanisms should be preferred; however, the release of drugs from nanoparticles still requires further investigations. PMID:21919898

  14. Selection of Nanobodies that Target Human Neonatal Fc Receptor

    PubMed Central

    Andersen, Jan Terje; Gonzalez-Pajuelo, Maria; Foss, Stian; Landsverk, Ole J. B.; Pinto, Débora; Szyroki, Alexander; de Haard, Hans J.; Saunders, Michael; Vanlandschoot, Peter; Sandlie, Inger

    2013-01-01

    FcRn is a key player in several immunological and non-immunological processes, as it mediates maternal-fetal transfer of IgG, regulates the serum persistence of IgG and albumin, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation. Thus, there is an intense interest in studies of how FcRn binds and transports its cargo within and across several types of cells, and FcRn detection reagents are in high demand. Here we report on phage display-selected Nanobodies that target human FcRn. The Nanobodies were obtained from a variable-domain repertoire library isolated from a llama immunized with recombinant human FcRn. One candidate, Nb218-H4, was shown to bind FcRn with high affinity at both acidic and neutral pH, without competing ligand binding and interfering with FcRn functions, such as transcytosis of IgG. Thus, Nb218-H4 can be used as a detection probe and as a tracker for visualization of FcRn-mediated cellular transport. PMID:23346375

  15. Caenorhabditis elegans as a model for lysosomal storage disorders.

    PubMed

    de Voer, Gert; Peters, Dorien; Taschner, Peter E M

    2008-01-01

    The nematode Caenorhabditis elegans is the simplest animal model available to study human disease. In this review, the worm homologues for the 58 human genes involved in lysosomal storage disorders and for 105 human genes associated with lysosomal function have been compiled. Most human genes had at least one worm homologue. In addition, the phenotypes of 147 mutants, in which these genes have been disrupted or knocked down, have been summarized and discussed. The phenotypic spectrum of worm models of lysosomal storage disorders varies from lethality to none obvious, with a large variety of intermediate phenotypes. The genetic power of C. elegans provides a means to identify genes involved in specific processes with relative ease. The overview of potential lysosomal phenotypes presented here might be used as a starting point for the phenotypic characterization of newly developed knock-out models or for the design of genetic screens selecting for loss or gain of suitable knock-out model phenotypes. Screens for genes involved in lysosomal biogenesis and function have been performed successfully resulting in the cup and glo mutants, but screens involving subtle phenotypes are likely to be difficult. PMID:18501720

  16. Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models

    PubMed Central

    Butler, David; Hwang, Jeannie; Estick, Candice; Nishiyama, Akiko; Kumar, Saranya Santhosh; Baveghems, Clive; Young-Oxendine, Hollie B.; Wisniewski, Meagan L.; Charalambides, Ana; Bahr, Ben A.

    2011-01-01

    Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid ? peptide (A?) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including A?142. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of A? pathology. Systemic PADK injections in APPSwInd and APPswe/PS1?E9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both A? immunostaining as well as A?x-42 sandwich ELISA measures in APPSwInd mice of 1011 months. More extensive A? deposition in 20-22-month APPswe/PS1?E9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic A?138 occurs as A?142 levels decrease in the mouse models, indicating that PADK treatment leads to A? truncation. Associated with A? clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces A?142 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of A? pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders. PMID:21695208

  17. Input Control Processes in Rapid Serial Visual Presentations: Target Selection and Distractor Inhibition

    ERIC Educational Resources Information Center

    Olivers, Christian N. L.; Watson, Derrick G.

    2006-01-01

    The attentional blink refers to the finding that the 2nd of 2 targets embedded in a stream of rapidly presented distractors is often missed. Whereas most theories of the attentional blink focus on limited-capacity processes that occur after target selection, the present work investigates the selection process itself. Identifying a target letter…

  18. Input Control Processes in Rapid Serial Visual Presentations: Target Selection and Distractor Inhibition

    ERIC Educational Resources Information Center

    Olivers, Christian N. L.; Watson, Derrick G.

    2006-01-01

    The attentional blink refers to the finding that the 2nd of 2 targets embedded in a stream of rapidly presented distractors is often missed. Whereas most theories of the attentional blink focus on limited-capacity processes that occur after target selection, the present work investigates the selection process itself. Identifying a target letter

  19. Autophagy negatively regulates cancer cell proliferation via selectively targeting VPRBP.

    PubMed

    Wang, Bo-Shi; Liu, Yi-Zhen; Yang, Yang; Zhang, Yu; Hao, Jia-Jie; Yang, Hai; Wang, Xiao-Min; Zhang, Zi-Qiang; Zhan, Qi-Min; Wang, Ming-Rong

    2013-02-01

    There have been multiple lines of evidence suggesting that autophagy selectively targets signalling proteins and regulates cancer cell signalling in addition to bulk clearance of long-lived proteins and organelles. Protein degradation through autophagy requires receptor protein LC3B to sequester the substrates into the autophagosome. In the present study, we screened LC3B (light-chain 3B)-binding partners and identified autophagic substrates in cancer cells. With lung cancer NCI-H1975 and oesophageal cancer KYSE30 cell lines as models, we found that VPRBP (viral protein R-binding protein) was a novel LC3B-binding protein through GST (glutathione transferase)-LC3B pull-down combined with LC-MS/MS (liquid chromatography-tandem MS) methods. Co-immunoprecipitation assay showed that VPRBP-LC3/p62 were in the same protein complex as the two cell lines. Induction of autophagy led to a down-regulation of VPRPB, which could be rescued by the inhibition of autophagy degradation by BFA1 (bafilomycin A1) and by the disruption of autophagy through ATG5-knockdown. We also found that induction of autophagy promotes VPRBP-LC3/p62 interaction. Immunohistochemical examination of human NSCLC (non-small cell lung cancer) tissues showed that VPRBP was positively correlated with p62 and negatively correlated with LC3B. Moreover, p62 and VPRBP were associated with poor prognosis in lung ADC (adenocarcinoma) (p62, P=0.019; VPRBP, P=0.005). Patients with low expression of both p62 and VPRBP showed the best prognosis. PMID:22963397

  20. Target product selection - where can Molecular Pharming make the difference?

    PubMed

    Paul, Mathew J; Teh, Audrey Y H; Twyman, Richard M; Ma, Julian K-C

    2013-01-01

    Four major developments have taken place in the world of Molecular Pharming recently. In the USA, the DARPA initiative challenged plant biotechnology companies to develop strategies for the large-scale manufacture of influenza vaccines, resulting in a successful Phase I clinical trial; in Europe the Pharma-Planta academic consortium gained regulatory approval for a plant-derived monoclonal antibody and completed a first-in-human phase I clinical trial; the Dutch pharmaceutical company Synthon acquired the assets of Biolex Therapeutics, an established Molecular Pharming company with several clinical candidates produced in their proprietary LEX system based on aquatic plants; and finally, the Israeli biotechnology company Protalix Biotherapeutics won FDA approval for the commercial release of a recombinant form of the enzyme glucocerebrosidase produced in carrot cells, the first plant biotechnology-derived biopharmaceutical in the world approved for the market. Commercial momentum is gathering pace with additional candidates now undergoing or awaiting approval for phase III clinical trials. Filling the product pipeline is vital to establish commercial sustainability, and the selection of appropriate target products for Molecular Pharming will be a critical factor. An interesting feature of the four stories outlined above is that they span the use of very different platform technologies addressing different types of molecules which aim to satisfy distinct market demands. In each case, Molecular Pharming was an economically and technically suitable approach, but this decisionmaking process is not necessarily straightforward. Although the various technologies available to Molecular Pharming are broad ranging and flexible, competing technologies are better established, so there needs to be a compelling reason to move into plants. It is most unlikely that plant biotechnology will be the answer for the whole biologics field. In this article, we discuss the current plant biotechnology approaches that appear to hold the greatest promise and in doing so attempt to define the product areas that are most likely to benefit from different Molecular Pharming technologies. PMID:23394563

  1. Potential Pitfalls and Solutions for Use of Fluorescent Fusion Proteins to Study the Lysosome

    PubMed Central

    Huang, Ling; Pike, Douglas; Sleat, David E.; Nanda, Vikas; Lobel, Peter

    2014-01-01

    Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications. PMID:24586430

  2. A shortcut to the lysosome: the mannose-6-phosphate-independent pathway.

    PubMed

    Coutinho, Maria Francisca; Prata, Maria Joo; Alves, Sandra

    2012-11-01

    Lysosomal hydrolases have long been known to be responsible for the degradation of different substrates in the cell. These acid hydrolases are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the trans-Golgi network (TGN). From there, they are delivered to endosomal/lysosomal compartments, where they finally become active due to the acidic pH characteristic of the lysosomal compartment. The majority of the enzymes leave the TGN after modification with mannose-6-phosphate (M6P) residues, which are specifically recognized by M6P receptors (MPRs), ensuring their transport to the endosomal/lysosomal system. Although M6P receptors play a major role in the intracellular transport of newly synthesized lysosomal enzymes in mammalian cells, several lines of evidence suggest the existence of alternative processes of lysosomal targeting. Among them, the two that are mediated by the M6P alternative receptors, lysosomal integral membrane protein (LIMP-2) and sortilin, have gained unequivocal support. LIMP-2 was shown to be implicated in the delivery of beta-glucocerebrosidase (GCase) to the lysosomes, whereas sortilin has been suggested to be a multifunctional receptor capable of binding several different ligands, including neurotensin and receptor-associated protein (RAP), and of targeting several proteins to the lysosome, including sphingolipid activator proteins (prosaposin and GM2 activator protein), acid sphingomyelinase and cathepsins D and H. Here, we review the current knowledge on these two proteins: their discovery, study, structural features and cellular function, with special attention to their role as alternative receptors to lysosomal trafficking. Recent studies associating both LIMP2 and sortilin to disease are also extensively reviewed. PMID:22884962

  3. Lysosomes and the plasma membrane

    PubMed Central

    Andrews, Norma W.

    2002-01-01

    Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury. PMID:12147679

  4. Controlling nematodes in dairy calves using targeted selective treatments.

    PubMed

    O'Shaughnessy, J; Earley, B; Mee, J F; Doherty, M L; Crosson, P; Barrett, D; de Waal, T

    2015-04-30

    With increasing concerns of anthelmintic resistance in cattle nematode populations worldwide, there is a need to explore alternative approaches to nematode control. One alternative approach is the use of targeted selective treatments (TST) where only individual animals are treated instead of the entire group. This study reports the findings of a TST approach in dairy calves conducted over their first grazing season (FGS) to control both gastrointestinal nematode and lungworm challenge. Ninety-six calves with an initial mean (s.d.) age and live weight of 130 (28.3) days and 120 (23.6)kg, respectively, were randomised by breed, age and live weight to one of two treatments; Control (n=24; 2) and TST (n=24; 2). Control calves were treated three times at pasture with ivermectin by subcutaneous injection. Individual calves in the TST group were treated at pasture with ivermectin when one of the following thresholds was met: (1) positive for lungworm larvae using the modified Baermann technique or (2) positive or negative for lungworm larvae using the modified Baermann technique with plasma pepsinogen concentration (PP) ? two international units of tyrosine/litre and faecal egg count (FEC) ? 200 strongyle eggs per gram of faeces. Calves were rotationally grazed from July 3rd 2012 (day 0) to November 2nd 2012 (day 122) when calves were housed. Calves were weighed and sampled (blood and faecal) every three weeks. There was an effect of treatment and time on both FEC [treatment (P=0.023), time (P<0.001)] and PP [treatment (P=0.002), time (P<0.001)]. Both FEC and PP were higher in TST calves. There was a 50% reduction in anthelmintic use in TST calves compared to control calves. Clinical signs of lungworm infection, confirmed by the modified Baermann technique, were evident in TST calves on days 62 and 63 of the study. The average daily live weight gain for control and TST calves was 0.50 (0.02)kg day(-1) and 0.47 (0.03)kg day(-1), respectively (P=0.41). Thus, performance in dairy calves can potentially be maintained with fewer anthelmintic treatments but farmers need to be vigilant of the challenge posed by lungworm. Any future approach into the use of TST in FGS calves must take into consideration the relative importance of lungworm as a pathogen. PMID:25770853

  5. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins.

    PubMed

    McEwan, David G; Popovic, Doris; Gubas, Andrea; Terawaki, Seigo; Suzuki, Hironori; Stadel, Daniela; Coxon, Fraser P; Miranda de Stegmann, Diana; Bhogaraju, Sagar; Maddi, Karthik; Kirchof, Anja; Gatti, Evelina; Helfrich, Miep H; Wakatsuki, Soichi; Behrends, Christian; Pierre, Philippe; Dikic, Ivan

    2015-01-01

    The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways. PMID:25498145

  6. Leaving the lysosome behind: novel developments in autophagy inhibition.

    PubMed

    Solitro, Abigail R; MacKeigan, Jeffrey P

    2016-01-01

    The search for a single silver bullet for the treatment of cancer has now been overshadowed by the identification of multiple therapeutic targets unique to each malignancy and even to each patient. In recent years, autophagy has emerged as one such therapeutic target. In response to both therapeutic and oncogenic stress, cancer cells upregulate and demonstrate an increased dependence upon this intracellular recycling process. Particularly in malignancies that currently lack targeted therapeutic options, autophagy inhibitors are the next hopeful prospects for the treatment of this disease. In this review, we discuss the rapid evolution of autophagy inhibitors from early lysosomotropic agents to next-generation lysosome-targeted drugs and beyond. PMID:26689099

  7. A determination of the steady state lysosomal pH of bloodstream stage African trypanosomes.

    PubMed

    McCann, Amanda K; Schwartz, Kevin J; Bangs, James D

    2008-06-01

    The lysosomal/endosomal system of African trypanosomes is developmentally regulated and is important in the pathogenesis associated with infection of the mammalian bloodstream. Long considered to be a target for drug development, the internal pH of the lysosome has been variously reported to range from <5.0 to >6.0. We have refined a flow cytometric technique using a pH-sensitive probe that specifically targets the lysosome, tomato lectin:Oregon Green 488 conjugate. The probe is delivered to the lysosome with fidelity, where it is shielded against external pH. Measurement of fluorescent output in the presence and absence of lysomotropic agent (NH(4)Cl) then allows precise titration of steady state lysosomal pH (4.84+/-0.23). Using bafilomycin A1 to inhibit acidification we demonstrate that this method is responsive to pharmacological perturbation of lysosomal physiology. This work should facilitate future studies of the lysosomal function in African trypanosomiasis, as well as other parasitic protozoa. PMID:18359105

  8. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

    PubMed

    Seebacher, Nicole A; Lane, Darius J R; Jansson, Patric J; Richardson, Des R

    2016-02-19

    Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in multidrug-resistant tumors where a stressful micro-environment exists. PMID:26601947

  9. Dynamin Regulates Autophagy by Modulating Lysosomal Function.

    PubMed

    Fang, Xuefei; Zhou, Jia; Liu, Wei; Duan, Xiuying; Gala, Upasana; Sandoval, Hector; Jaiswal, Manish; Tong, Chao

    2016-02-20

    Autophagy is a central lysosomal degradation pathway required for maintaining cellular homeostasis and its dysfunction is associated with numerous human diseases. To identify players in autophagy, we tested ∼1200 chemically induced mutations on the X chromosome in Drosophila fat body clones and discovered that shibire (shi) plays an essential role in starvation-induced autophagy. shi encodes a dynamin protein required for fission of clathrin-coated vesicles from the plasma membrane during endocytosis. We showed that Shi is dispensable for autophagy initiation and autophagosome-lysosome fusion, but required for lysosomal/autolysosomal acidification. We also showed that other endocytic core machinery components like clathrin and AP2 play similar but not identical roles in regulating autophagy and lysosomal function as dynamin. Previous studies suggested that dynamin directly regulates autophagosome formation and autophagic lysosome reformation (ALR) through its excision activity. Here, we provide evidence that dynamin also regulates autophagy indirectly by regulating lysosomal function. PMID:26924690

  10. Genes Associated with SLE Are Targets of Recent Positive Selection

    PubMed Central

    Ramos, Paula S.; Shaftman, Stephanie R.; Ward, Ralph C.; Langefeld, Carl D.

    2014-01-01

    The reasons for the ethnic disparities in the prevalence of systemic lupus erythematosus (SLE) and the relative high frequency of SLE risk alleles in the population are not fully understood. Population genetic factors such as natural selection alter allele frequencies over generations and may help explain the persistence of such common risk variants in the population and the differential risk of SLE. In order to better understand the genetic basis of SLE that might be due to natural selection, a total of 74 genomic regions with compelling evidence for association with SLE were tested for evidence of recent positive selection in the HapMap and HGDP populations, using population differentiation, allele frequency, and haplotype-based tests. Consistent signs of positive selection across different studies and statistical methods were observed at several SLE-associated loci, including PTPN22, TNFSF4, TET3-DGUOK, TNIP1, UHRF1BP1, BLK, and ITGAM genes. This study is the first to evaluate and report that several SLE-associated regions show signs of positive natural selection. These results provide corroborating evidence in support of recent positive selection as one mechanism underlying the elevated population frequency of SLE risk loci and supports future research that integrates signals of natural selection to help identify functional SLE risk alleles. PMID:24587899

  11. Evaluating Gaze-Based Interface Tools to Facilitate Point-and-Select Tasks with Small Targets

    ERIC Educational Resources Information Center

    Skovsgaard, Henrik; Mateo, Julio C.; Hansen, John Paulin

    2011-01-01

    Gaze interaction affords hands-free control of computers. Pointing to and selecting small targets using gaze alone is difficult because of the limited accuracy of gaze pointing. This is the first experimental comparison of gaze-based interface tools for small-target (e.g. less than 12 x 12 pixels) point-and-select tasks. We conducted two

  12. From rare to common and back again: 60years of lysosomal dysfunction.

    PubMed

    Coutinho, Maria Francisca; Alves, Sandra

    2016-02-01

    Sixty years after its discovery, the lysosome is no longer considered as cell's waste bin but as an organelle playing a central role in cell metabolism. Besides its well known association with lysosomal storage disorders (mostly rare and life-threatening diseases), recent data have shown that the lysosome is also a player in some of the most common conditions of our time; and, perhaps even most important, it is not only a target for orphan drugs (rare disease therapeutic approaches) but also a putative target to treat patients suffering from common complex diseases worldwide. Here we review the striking associations linking rare lysosomal storage disorders such as the well-known Gaucher disease, or even the recently discovered, extremely rare Neuronal Ceroid Lipofuscinosis-11 and some of the most frequent, multifaceted and complex disorders of modern society such as cancer, Parkinson's disease and frontotemporal lobar degeneration. PMID:26422115

  13. Parasite neuropeptide biology: Seeding rational drug target selection?

    PubMed Central

    McVeigh, Paul; Atkinson, Louise; Marks, Nikki J.; Mousley, Angela; Dalzell, Johnathan J.; Sluder, Ann; Hammerland, Lance; Maule, Aaron G.

    2011-01-01

    The rationale for identifying drug targets within helminth neuromuscular signalling systems is based on the premise that adequate nerve and muscle function is essential for many of the key behavioural determinants of helminth parasitism, including sensory perception/host location, invasion, locomotion/orientation, attachment, feeding and reproduction. This premise is validated by the tendency of current anthelmintics to act on classical neurotransmitter-gated ion channels present on helminth nerve and/or muscle, yielding therapeutic endpoints associated with paralysis and/or death. Supplementary to classical neurotransmitters, helminth nervous systems are peptide-rich and encompass associated biosynthetic and signal transduction components – putative drug targets that remain to be exploited by anthelmintic chemotherapy. At this time, no neuropeptide system-targeting lead compounds have been reported, and given that our basic knowledge of neuropeptide biology in parasitic helminths remains inadequate, the short-term prospects for such drugs remain poor. Here, we review current knowledge of neuropeptide signalling in Nematoda and Platyhelminthes, and highlight a suite of 19 protein families that yield deleterious phenotypes in helminth reverse genetics screens. We suggest that orthologues of some of these peptidergic signalling components represent appealing therapeutic targets in parasitic helminths. PMID:24533265

  14. Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid Lipase and Potential Niemann-Pick Type C Disease Therapeuticsa

    PubMed Central

    Rosenbaum, Anton I.; Cosner, Casey C.; Mariani, Christopher J.; Maxfield, Frederick R.; Wiest, Olaf; Helquist, Paul

    2010-01-01

    Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat. PMID:20557099

  15. Regional Personalized Electrodes to Select Transcranial Current Stimulation Target

    PubMed Central

    Tecchio, Franca; Cancelli, A.; Cottone, C.; Tomasevic, L.; Devigus, B.; Zito, G.; Ercolani, Matilde; Carducci, F.

    2013-01-01

    Rationale: Personalizing transcranial stimulations promises to enhance beneficial effects for individual patients. Objective: To stimulate specific cortical regions by developing a procedure to bend and position custom shaped electrodes; to probe the effects on cortical excitability produced when the properly customized electrode is targeting different cortical areas. Method: An ad hoc neuronavigation procedure was developed to accurately shape and place the personalized electrodes on the basis of individual brain magnetic resonance images (MRI) on bilateral primary motor (M1) and somatosensory (S1) cortices. The transcranial alternating current stimulation (tACS) protocol published by Feurra et al. (2011b) was used to test the effects on cortical excitability of the personalized electrode when targeting S1 or M1. Results: Neuronal excitability as evaluated by tACS was different when targeting M1 or S1, with the General Estimating Equation model indicating a clear tCS Effect (p?targeted by tCS properly shaping and positioning the stimulating electrode. Significance: Through multimodal brain investigations continuous efforts in understanding the neuronal changes related to specific neurological or psychiatric diseases become more relevant as our ability to build the compensating interventions improves. An important step forward on this path is the ability to target the specific cortical area of interest, as shown in the present pilot work. PMID:23626529

  16. TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function.

    PubMed

    Cang, Chunlei; Aranda, Kimberly; Seo, Young-jun; Gasnier, Bruno; Ren, Dejian

    2015-08-27

    Potassium is the most abundant ion to face bothplasma and organelle membranes. Extensive research over the past seven decades has characterized how K(+) permeates the plasma membrane to control fundamental processes such as secretion, neuronal communication, and heartbeat. However, how K(+) permeates organelles such as lysosomes and endosomes is unknown. Here, we directly recorded organelle K(+) conductance and discovered a major K(+)-selective channel KEL on endosomes and lysosomes. KEL is formed by TMEM175, a protein with unknown function. Unlike any of the ?80 plasma membrane K(+) channels, TMEM175 has two repeats of 6-transmembrane-spanning segments and has no GYG K(+) channel sequence signature-containing, pore-forming P loop. Lysosomes lacking TMEM175 exhibit no K(+) conductance, have a markedly depolarized ?? and little sensitivity to changes in [K(+)], andhave compromised luminal pH stability and abnormal fusion with autophagosomes during autophagy. Thus, TMEM175 comprises a K(+) channel that underlies the molecular mechanism of lysosomal K(+) permeability. PMID:26317472

  17. Genome-wide polymorphisms show unexpected targets of natural selection.

    PubMed

    Pespeni, Melissa H; Garfield, David A; Manier, Mollie K; Palumbi, Stephen R

    2012-04-01

    Natural selection can act on all the expressed genes of an individual, leaving signatures of genetic differentiation or diversity at many loci across the genome. New power to assay these genome-wide effects of selection comes from associating multi-locus patterns of polymorphism with gene expression and function. Here, we performed one of the first genome-wide surveys in a marine species, comparing purple sea urchins, Strongylocentrotus purpuratus, from two distant locations along the species' wide latitudinal range. We examined 9112 polymorphic loci from upstream non-coding and coding regions of genes for signatures of selection with respect to gene function and tissue- and ontogenetic gene expression. We found that genetic differentiation (F(ST)) varied significantly across functional gene classes. The strongest enrichment occurred in the upstream regions of E3 ligase genes, enzymes known to regulate protein abundance during development and environmental stress. We found enrichment for high heterozygosity in genes directly involved in immune response, particularly NALP genes, which mediate pro-inflammatory signals during bacterial infection. We also found higher heterozygosity in immune genes in the southern population, where disease incidence and pathogen diversity are greater. Similar to the major histocompatibility complex in mammals, balancing selection may enhance genetic diversity in the innate immune system genes of this invertebrate. Overall, our results show that how genome-wide polymorphism data coupled with growing databases on gene function and expression can combine to detect otherwise hidden signals of selection in natural populations. PMID:21993504

  18. Genome-wide polymorphisms show unexpected targets of natural selection

    PubMed Central

    Pespeni, Melissa H.; Garfield, David A.; Manier, Mollie K.; Palumbi, Stephen R.

    2012-01-01

    Natural selection can act on all the expressed genes of an individual, leaving signatures of genetic differentiation or diversity at many loci across the genome. New power to assay these genome-wide effects of selection comes from associating multi-locus patterns of polymorphism with gene expression and function. Here, we performed one of the first genome-wide surveys in a marine species, comparing purple sea urchins, Strongylocentrotus purpuratus, from two distant locations along the species' wide latitudinal range. We examined 9112 polymorphic loci from upstream non-coding and coding regions of genes for signatures of selection with respect to gene function and tissue- and ontogenetic gene expression. We found that genetic differentiation (FST) varied significantly across functional gene classes. The strongest enrichment occurred in the upstream regions of E3 ligase genes, enzymes known to regulate protein abundance during development and environmental stress. We found enrichment for high heterozygosity in genes directly involved in immune response, particularly NALP genes, which mediate pro-inflammatory signals during bacterial infection. We also found higher heterozygosity in immune genes in the southern population, where disease incidence and pathogen diversity are greater. Similar to the major histocompatibility complex in mammals, balancing selection may enhance genetic diversity in the innate immune system genes of this invertebrate. Overall, our results show that how genome-wide polymorphism data coupled with growing databases on gene function and expression can combine to detect otherwise hidden signals of selection in natural populations. PMID:21993504

  19. Acid phosphatase 5 is responsible for removing the mannose 6-phosphate recognition marker from lysosomal proteins

    PubMed Central

    Sun, Pengling; Sleat, David E.; Lecocq, Michle; Hayman, Alison R.; Jadot, Michel; Lobel, Peter

    2008-01-01

    Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine. PMID:18940929

  20. Apolipoprotein L-I Promotes Trypanosome Lysis by Forming Pores in Lysosomal Membranes

    NASA Astrophysics Data System (ADS)

    Pérez-Morga, David; Vanhollebeke, Benoit; Paturiaux-Hanocq, Françoise; Nolan, Derek P.; Lins, Laurence; Homblé, Fabrice; Vanhamme, Luc; Tebabi, Patricia; Pays, Annette; Poelvoorde, Philippe; Jacquet, Alain; Brasseur, Robert; Pays, Etienne

    2005-07-01

    Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that this protein contains a membrane pore-forming domain functionally similar to that of bacterial colicins, flanked by a membrane-addressing domain. In lipid bilayer membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei, apolipoprotein L-I was targeted to the lysosomal membrane and triggered depolarization of this membrane, continuous influx of chloride, and subsequent osmotic swelling of the lysosome until the trypanosome lysed.

  1. The safety of ONRAB in select non-target wildlife.

    TOXLINE Toxicology Bibliographic Information

    Fry TL; Vandalen KK; Duncan C; Vercauteren K

    2013-08-20

    ONRAB() is a recombinant human adenovirus type 5 (HAd5) with the rabies glycoprotein gene incorporated into its genome. ONRAB() has been used in Canada as an oral rabies vaccine in target wildlife species such as: red fox (Vulpes vulpes), raccoon (Procyon lotor), and striped skunk (Mepthis mephitis). We evaluated the safety of ONRAB() in non-target wildlife species likely to contact the vaccine baits during oral rabies vaccine campaigns in the United States. We investigated the effects of oral inoculation of high titer ONRAB(), approximately ten times the dose given to target species, in wood rats (Neotoma spp.), eastern cottontail rabbits (Sylvilagus floridanus), Virginia opossums (Didelphis virginiana), eastern wild turkeys (Meleagris gallopavo silvestri), and fox squirrels (Sciurus niger). We performed real-time polymerase chain reaction (PCR) on fecal swabs, oral swabs, and tissues, including lung, liver, kidney, small intestine, large intestine, and when appropriate nasal turbinates, to detect ONRAB() DNA from inoculated animals. By seven days post-inoculation, turkeys, opossums, and cottontails had all stopped shedding ONRAB() DNA. One wood rat and one fox squirrel still had detectable levels of ONRAB() DNA in fecal swabs 14 days post-inoculation. Real-time PCR analysis of the tissues revealed some ONRAB() DNA persisting in certain tissues; however, there were no significant gross or histologic lesions associated with ONRAB() in any of the species studied. Our results suggest that many non-target species are not likely to be impacted by the distribution of ONRAB() as part of oral rabies vaccination programs in the United States.

  2. The safety of ONRAB in select non-target wildlife.

    PubMed

    Fry, Tricia L; Vandalen, Kaci K; Duncan, Colleen; Vercauteren, Kurt

    2013-08-20

    ONRAB() is a recombinant human adenovirus type 5 (HAd5) with the rabies glycoprotein gene incorporated into its genome. ONRAB() has been used in Canada as an oral rabies vaccine in target wildlife species such as: red fox (Vulpes vulpes), raccoon (Procyon lotor), and striped skunk (Mepthis mephitis). We evaluated the safety of ONRAB() in non-target wildlife species likely to contact the vaccine baits during oral rabies vaccine campaigns in the United States. We investigated the effects of oral inoculation of high titer ONRAB(), approximately ten times the dose given to target species, in wood rats (Neotoma spp.), eastern cottontail rabbits (Sylvilagus floridanus), Virginia opossums (Didelphis virginiana), eastern wild turkeys (Meleagris gallopavo silvestri), and fox squirrels (Sciurus niger). We performed real-time polymerase chain reaction (PCR) on fecal swabs, oral swabs, and tissues, including lung, liver, kidney, small intestine, large intestine, and when appropriate nasal turbinates, to detect ONRAB() DNA from inoculated animals. By seven days post-inoculation, turkeys, opossums, and cottontails had all stopped shedding ONRAB() DNA. One wood rat and one fox squirrel still had detectable levels of ONRAB() DNA in fecal swabs 14 days post-inoculation. Real-time PCR analysis of the tissues revealed some ONRAB() DNA persisting in certain tissues; however, there were no significant gross or histologic lesions associated with ONRAB() in any of the species studied. Our results suggest that many non-target species are not likely to be impacted by the distribution of ONRAB() as part of oral rabies vaccination programs in the United States. PMID:23831321

  3. Selective imaging of adherent targeted ultrasound contrast agents

    NASA Astrophysics Data System (ADS)

    Zhao, S.; Kruse, D. E.; Ferrara, K. W.; Dayton, P. A.

    2007-04-01

    The goal of ultrasonic molecular imaging is the detection of targeted contrast agents bound to receptors on endothelial cells. We propose imaging methods that can distinguish adherent microbubbles from tissue and from freely circulating microbubbles, each of which would otherwise obscure signal from molecularly targeted adherent agents. The methods are based on a harmonic signal model of the returned echoes over a train of pulses. The first method utilizes an 'image-push-image' pulse sequence where adhesion of contrast agents is rapidly promoted by acoustic radiation force and the presence of adherent agents is detected by the signal change due to targeted microbubble adhesion. The second method rejects tissue echoes using a spectral high-pass filter. Free agent signal is suppressed by a pulse-to-pulse low-pass filter in both methods. An overlay of the adherent and/or flowing contrast agents on B-mode images can be readily created for anatomical reference. Contrast-to-tissue ratios from adherent microbubbles exceeding 30 dB and 20 dB were achieved for the two methods proposed, respectively. The performance of these algorithms is compared, emphasizing the significance and potential applications in ultrasonic molecular imaging.

  4. Target selection by natural and redesigned PUF proteins.

    PubMed

    Porter, Douglas F; Koh, Yvonne Y; VanVeller, Brett; Raines, Ronald T; Wickens, Marvin

    2015-12-29

    Pumilio/fem-3 mRNA binding factor (PUF) proteins bind RNA with sequence specificity and modularity, and have become exemplary scaffolds in the reengineering of new RNA specificities. Here, we report the in vivo RNA binding sites of wild-type (WT) and reengineered forms of the PUF protein Saccharomyces cerevisiae Puf2p across the transcriptome. Puf2p defines an ancient protein family present throughout fungi, with divergent and distinctive PUF RNA binding domains, RNA-recognition motifs (RRMs), and prion regions. We identify sites in RNA bound to Puf2p in vivo by using two forms of UV cross-linking followed by immunopurification. The protein specifically binds more than 1,000 mRNAs, which contain multiple iterations of UAAU-binding elements. Regions outside the PUF domain, including the RRM, enhance discrimination among targets. Compensatory mutants reveal that one Puf2p molecule binds one UAAU sequence, and align the protein with the RNA site. Based on this architecture, we redesign Puf2p to bind UAAG and identify the targets of this reengineered PUF in vivo. The mutant protein finds its target site in 1,800 RNAs and yields a novel RNA network with a dramatic redistribution of binding elements. The mutant protein exhibits even greater RNA specificity than wild type. The redesigned protein decreases the abundance of RNAs in its redesigned network. These results suggest that reengineering using the PUF scaffold redirects and can even enhance specificity in vivo. PMID:26668354

  5. A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues

    NASA Astrophysics Data System (ADS)

    Fan, Jiangli; Han, Zhichao; Kang, Yao; Peng, Xiaojun

    2016-01-01

    Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy.

  6. A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues.

    PubMed

    Fan, Jiangli; Han, Zhichao; Kang, Yao; Peng, Xiaojun

    2016-01-01

    Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy. PMID:26794434

  7. A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues

    PubMed Central

    Fan, Jiangli; Han, Zhichao; Kang, Yao; Peng, Xiaojun

    2016-01-01

    Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy. PMID:26794434

  8. Signal Transduction and Molecular Targets of Selected Flavonoids

    PubMed Central

    Bode, Ann M.

    2013-01-01

    Abstract Significance: Diet exerts a major influence on the risk for developing cancer and heart disease. Food factors such as flavonoids are alleged to protect cells from premature aging and disease by shielding DNA, proteins, and lipids from oxidative damage. Recent Advances: Our work has focused on clarifying the effects of dietary components on cancer cell proliferation and tumor growth, discovering mechanisms to explain the effects, and identifying the specific molecular targets of these compounds. Our strategy for identifying specific molecular targets of phytochemicals involves the use of supercomputer technology combined with protein crystallography, molecular biology, and experimental laboratory verification. Critical Issues: One of the greatest challenges for scientists is to reduce the accumulation of distortion and half truths reported in the popular media regarding the health benefits of certain foods or food supplements. The use of these is not new, but interest has increased dramatically because of perceived health benefits that are presumably acquired without unpleasant side effects. Flavonoids are touted to exert many beneficial effects in vitro. However, whether they can produce these effects in vivo is disputed. Future Directions: The World Health Organization indicates that one third of all cancer deaths are preventable and that diet is closely linked to prevention. Based on this idea and epidemiological findings, attention has centered on dietary phytochemicals as an effective intervention in cancer development. However, an unequivocal link between diet and cancer has not been established. Thus, identifying cancer preventive dietary agents with specific molecular targets is essential to move forward toward successful cancer prevention. Antioxid. Redox Signal. 19, 163–180. PMID:23458437

  9. Leveraging Big Data to Transform Target Selection and Drug Discovery

    PubMed Central

    Chen, B; Butte, AJ

    2016-01-01

    The advances of genomics, sequencing, and high throughput technologies have led to the creation of large volumes of diverse datasets for drug discovery. Analyzing these datasets to better understand disease and discover new drugs is becoming more common. Recent open data initiatives in basic and clinical research have dramatically increased the types of data available to the public. The past few years have witnessed successful use of big data in many sectors across the whole drug discovery pipeline. In this review, we will highlight the state of the art in leveraging big data to identify new targets, drug indications, and drug response biomarkers in this era of precision medicine. PMID:26659699

  10. Leveraging big data to transform target selection and drug discovery.

    PubMed

    Chen, B; Butte, A J

    2016-03-01

    The advances of genomics, sequencing, and high throughput technologies have led to the creation of large volumes of diverse datasets for drug discovery. Analyzing these datasets to better understand disease and discover new drugs is becoming more common. Recent open data initiatives in basic and clinical research have dramatically increased the types of data available to the public. The past few years have witnessed successful use of big data in many sectors across the whole drug discovery pipeline. In this review, we will highlight the state of the art in leveraging big data to identify new targets, drug indications, and drug response biomarkers in this era of precision medicine. PMID:26659699

  11. Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

    PubMed

    Lee, Eric A; Angka, Leonard; Rota, Sarah-Grace; Hanlon, Thomas; Mitchell, Andrew; Hurren, Rose; Wang, Xiao Ming; Gronda, Marcela; Boyaci, Ezel; Bojko, Barbara; Minden, Mark; Sriskanthadevan, Shrivani; Datti, Alessandro; Wrana, Jeffery L; Edginton, Andrea; Pawliszyn, Janusz; Joseph, Jamie W; Quadrilatero, Joe; Schimmer, Aaron D; Spagnuolo, Paul A

    2015-06-15

    Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function. PMID:26077472

  12. Suppression of lysosome function induces autophagy via a feedback down-regulation of MTOR complex 1 (MTORC1) activity.

    PubMed

    Li, Min; Khambu, Bilon; Zhang, Hao; Kang, Jeong-Han; Chen, Xiaoyun; Chen, Daohong; Vollmer, Laura; Liu, Pei-Qing; Vogt, Andreas; Yin, Xiao-Ming

    2013-12-13

    Autophagy can be activated via MTORC1 down-regulation by amino acid deprivation and by certain chemicals such as rapamycin, torin, and niclosamide. Lysosome is the degrading machine for autophagy but has also been linked to MTORC1 activation through the Rag/RRAG GTPase pathway. This association raises the question of whether lysosome can be involved in the initiation of autophagy. Toward this end, we found that niclosamide, an MTORC1 inhibitor, was able to inhibit lysosome degradation and increase lysosomal permeability. Niclosamide was ineffective in inhibiting MTORC1 in cells expressing constitutively activated Rag proteins, suggesting that its inhibitory effects were targeted to the Rag-MTORC1 signaling system. This places niclosamide in the same category of bafilomycin A1 and concanamycin A, inhibitors of the vacuolar H(+)-ATPase, for its dependence on Rag GTPase in suppression of MTORC1. Surprisingly, classical lysosome inhibitors such as chloroquine, E64D, and pepstatin A were also able to inhibit MTORC1 in a Rag-dependent manner. These lysosome inhibitors were able to activate early autophagy events represented by ATG16L1 and ATG12 puncta formation. Our work established a link between the functional status of the lysosome in general to the Rag-MTORC1 signaling axis and autophagy activation. Thus, the lysosome is not only required for autophagic degradation but also affects autophagy activation. Lysosome inhibitors can have a dual effect in suppressing autophagy degradation and in initiating autophagy. PMID:24174532

  13. BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release.

    PubMed

    Cao, Qi; Zhong, Xi Zo; Zou, Yuanjie; Zhang, Zhu; Toro, Ligia; Dong, Xian-Ping

    2015-05-26

    Promoting lysosomal trafficking represents a promising therapeutic approach for lysosome storage diseases. Efficient Ca(2+) mobilization from lysosomes is important for lysosomal trafficking. Ca(2+) release from lysosomes could generate a negative potential in the lumen to disturb subsequent Ca(2+) release in the absence of counter ion flux. Here we report that lysosomes express big-conductance Ca(2+)-activated potassium (BK) channels that form physical and functional coupling with the lysosomal Ca(2+) release channel, TRPML1. Ca(2+) release via TRPML1 causes BK activation, which in turn facilitates further lysosomal Ca(2+) release and membrane trafficking. Importantly, BK overexpression rescues the impaired TRPML1-mediated Ca(2+) release and abnormal lysosomal storage in cells from Niemann-Pick C1 patients. Therefore, we have identified alysosomal K(+) channel that provides a positive feedback mechanism to facilitate TRPML1-mediated Ca(2+) release and membrane trafficking. Our findings suggest that upregulating BK may be a potential therapeutic strategy for certain lysosomal storage diseases and common neurodegenerative disorders. PMID:25982675

  14. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.

    PubMed

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P2-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation. PMID:26950892

  15. [Gesundheitsziele.de. Selection of a new health target for Germany].

    PubMed

    Maschewsky-Schneider, U; Goecke, M; Gocke, M; Hlscher, U; Kolip, P; Kuhn, A; Sewster, D; Zeeb, H

    2013-09-01

    In 2013, the forum gesundheitsziele.de selected "reduction of alcohol consumption" and "patient safety" as new health targets. Besides the two selected targets, three other topics were considered: health at work, health during pregnancy and childbirth, and health and migration. This paper describes the selection process, which followed several criteria: mortality, morbidity, prevalence, burden of disease, economic impact, potential for improvement, equity in health, empowerment of and priorities of health problems in the population. The analysis particularly focused on the assessment of the feasibility and the readiness of stakeholders to participate in the development and implementation of health targets. PMID:23990097

  16. Disulfiram-induced cytotoxicity and endo-lysosomal sequestration of zinc in breast cancer cells

    PubMed Central

    Wiggins, Helen L.; Wymant, Jennifer M.; Solfa, Francesca; Hiscox, Stephen E.; Taylor, Kathryn M.; Westwell, Andrew D.; Jones, Arwyn T.

    2015-01-01

    Disulfiram, a clinically used alcohol-deterrent has gained prominence as a potential anti-cancer agent due to its impact on copper-dependent processes. Few studies have investigated zinc effects on disulfiram action, despite it having high affinity for this metal. Here we studied the cytotoxic effects of disulfiram in breast cancer cells, and its relationship with both intra and extracellular zinc. MCF-7 and BT474 cancer cell lines gave a striking time-dependent biphasic cytotoxic response between 0.01 and 10?M disulfiram. Co-incubation of disulfiram with low-level zinc removed this effect, suggesting that availability of extracellular zinc significantly influences disulfiram efficacy. Live-cell confocal microscopy using fluorescent endocytic probes and the zinc dye Fluozin-3 revealed that disulfiram selectively and rapidly increased zinc levels in endo-lysosomes. Disulfiram also caused spatial disorganization of late endosomes and lysosomes, suggesting they are novel targets for this drug. This relationship between disulfiram toxicity and ionophore activity was consolidated via synthesis of a new disulfiram analog and overall we demonstrate a novel mechanism of disulfiram-cytotoxicity with significant clinical implications for future use as a cancer therapeutic. PMID:25557293

  17. Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes

    PubMed Central

    Carson, John M.; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B.; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin. PMID:24924335

  18. Podocytes degrade endocytosed albumin primarily in lysosomes.

    PubMed

    Carson, John M; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin. PMID:24924335

  19. Enzyme therapy for lysosomal acid lipase deficiency in the mouse.

    PubMed

    Du, H; Schiavi, S; Levine, M; Mishra, J; Heur, M; Grabowski, G A

    2001-08-01

    Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice. Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD. PMID:11487567

  20. Recent advances in gene therapy for lysosomal storage disorders

    PubMed Central

    Rastall, David PW; Amalfitano, Andrea

    2015-01-01

    Lysosomal storage disorders (LSDs) are a group of genetic diseases that result in metabolic derangements of the lysosome. Most LSDs are due to the genetic absence of a single catabolic enzyme, causing accumulation of the enzyme’s substrate within the lysosome. Over time, tissue-specific substrate accumulations result in a spectrum of symptoms and disabilities that vary by LSD. LSDs are promising targets for gene therapy because delivery of a single gene into a small percentage of the appropriate target cells may be sufficient to impact the clinical course of the disease. Recently, there have been several significant advancements in the potential for gene therapy of these disorders, including the first human trials. Future clinical trials will build upon these initial attempts, with an improved understanding of immune system responses to gene therapy, the obstacle that the blood–brain barrier poses for neuropathic LSDs, as well other biological barriers that, when overcome, may facilitate gene therapy for LSDs. In this manuscript, we will highlight the recent innovations in gene therapy for LSDs and discuss the clinical limitations that remain to be overcome, with the goal of fostering an understanding and further development of this important field. PMID:26170711

  1. Ceramide channel: Structural basis for selective membrane targeting.

    PubMed

    Perera, Meenu N; Ganesan, Vidyaramanan; Siskind, Leah J; Szulc, Zdzislaw M; Bielawska, Alicja; Bittman, Robert; Colombini, Marco

    2016-01-01

    A ceramide commonly found in mammalian cells, C16-ceramide (N-palmitoyl-d-erythro-sphingosine), is capable of forming large, protein-permeable channels in the mitochondrial outer membrane (MOM). However, C16-ceramide is unable to permeabilize the plasma membrane of erythrocytes. This specificity is unexpected considering that ceramide forms channels in simple phosphoglycerolipid membranes. Synthetic analogs of C16-ceramide with targeted changes at each of the functional regions of the molecule including methylation, altered hydrocarbon chain length, and changes in the stereochemistry, were tested to probe the role of ceramide's molecular features on its ability to form channels in these two different membrane types. The ability to permeabilize the MOM was relatively insensitive to modifications of the various functional groups of ceramide whereas the same modifications resulted in plasma membrane permeabilization (a gain of function rather than a loss of function). Some analogs (ceramine, NBD-labeled ceramide, C18,1 ceramide) gained another function, the ability to inhibit cytochrome oxidase. The gain of deleterious functions indicates that constraints on the structure of ceramide that is formed by the cell's synthetic machinery includes the avoidance of deleterious interactions. We propose that the specific structure of ceramide limits the size of its interactome (both proteins and lipids) thus reducing the likelihood of unwanted side effects. PMID:26408265

  2. Initial basalt target site selection evaluation for the Mars penetrator drop test

    NASA Technical Reports Server (NTRS)

    Bunch, T. E.; Quaide, W. L.; Polkowski, G.

    1976-01-01

    Potential basalt target sites for an air drop penetrator test were described and the criteria involved in site selection were discussed. A summary of the background field geology and recommendations for optimum sites are also presented.

  3. Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

    PubMed Central

    Oh, Doo-Byoung

    2015-01-01

    Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444] PMID:25999178

  4. Auditory Stream Segregation Improves Infants' Selective Attention to Target Tones Amid Distracters

    ERIC Educational Resources Information Center

    Smith, Nicholas A.; Trainor, Laurel J.

    2011-01-01

    This study examined the role of auditory stream segregation in the selective attention to target tones in infancy. Using a task adapted from Bregman and Rudnicky's 1975 study and implemented in a conditioned head-turn procedure, infant and adult listeners had to discriminate the temporal order of 2,200 and 2,400 Hz target tones presented alone,

  5. Joint Skewness and Its Application in Unsupervised Band Selection for Small Target Detection

    PubMed Central

    Geng, Xiurui; Sun, Kang; Ji, Luyan; Tang, Hairong; Zhao, Yongchao

    2015-01-01

    Few band selection methods are specially designed for small target detection. It is well known that the information of small targets is most likely contained in non-Gaussian bands, where small targets are more easily separated from the background. On the other hand, correlation of band set also plays an important role in the small target detection. When the selected bands are highly correlated, it will be unbeneficial for the subsequent detection. However, the existing non-Gaussianity-based band selection methods have not taken the correlation of bands into account, which generally result in high correlation of obtained bands. In this paper, combining the third-order (third-order tensor) and second-order (correlation) statistics of bands, we define a new concept, named joint skewness, for multivariate data. Moreover, we also propose an easy-to-implement approach to estimate this index based on high-order singular value decomposition (HOSVD). Based on the definition of joint skewness, we present an unsupervised band selection for small target detection for hyperspectral data, named joint skewness band selection (JSBS). The evaluation results demonstrate that the bands selected by JSBS are very effective in terms of small target detection. PMID:25873018

  6. The lh3 Glycosyltransferase Directs Target-Selective Peripheral Nerve Regeneration.

    PubMed

    Isaacman-Beck, Jesse; Schneider, Valerie; Franzini-Armstrong, Clara; Granato, Michael

    2015-11-18

    Functional PNS regeneration requires injured axons to return to their original synaptic targets, yet the mechanisms underlying target-selective regeneration have remained elusive. Using live-cell imaging in zebrafish we find that regenerating motor axons exhibit a strong preference for their original muscle territory and that axons probe both correct and incorrect trajectories extensively before selecting their original path. We show that this process requires the glycosyltransferase lh3 and that post-injury expression of lh3 in Schwann cells is sufficient to restore target-selective regeneration. Moreover, we demonstrate that Schwann cells neighboring the transection site express the lh3 substrate collagen4a5 and that during regeneration collagen4a5 destabilizes axons probing inappropriate trajectories to ensure target-selective regeneration, possibly through the axonal repellant slit1a. Our results demonstrate that selective ECM components match subpopulations of regenerating axons with their original targets and reveal a previously unappreciated mechanism that conveys synaptic target selection to regenerating axons invivo. VIDEO ABSTRACT. PMID:26549330

  7. Internalizing antibodies and targeted cancer therapy: direct selection from phage display libraries.

    PubMed

    Nielsen; Marks

    2000-08-01

    Antibody internalization is required for the success of many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for the targeted delivery of genes or viral DNA into cells. Recently, it has become possible to directly select antibody fragments from phage display libraries for internalization into mammalian cells. Here we review the therapeutic applications of internalized antibodies and describe how phage display enables the isolation of internalizing antibodies to novel or known targets. PMID:10916148

  8. Condition-dependent and condition-independent target selection in the macaque posterior parietal cortex.

    PubMed

    Ogawa, Tadashi; Komatsu, Hidehiko

    2009-02-01

    During a visual search, information about the visual attributes of an object and associated behavioral requirements is essential for discriminating a target object from others in the visual field. On the other hand, information about the object's position appears to be more important when orienting the eyes toward the target. To understand the neural mechanisms underlying such a transition (i.e., from nonspatial- to spatial-based target selection), we examined the dependence of neuronal activity in the macaque posterior parietal cortex (PPC) on visual sensory properties and ongoing task demands. Monkeys were trained to perform a visual search task in which either a shape or color singleton within an array was the target, depending on the ongoing search dimension. The visual properties and the task demands were manipulated by independently changing the stimulus features (shape and color), singleton type, and search dimension. We found that a subset of PPC neurons significantly discriminated the target from other stimuli only when the target was defined by a particular stimulus dimension and had specific stimulus features, such as a shape-singleton, bar stimulus (condition-dependent target selection), whereas another subset did so irrespective of the stimulus features and the target-defining dimension (condition-independent target selection). There was thus a great deal of variety in the neural representations specifying the locus of the target. The coexistence of these distinctly different types of target-discrimination processes suggests that the PPC may be situated at the level where the transition from nonspatial- to spatial-based target selection takes place. PMID:19073809

  9. SELECTION FOR ORCHARDGRASS SEED YIELD IN TARGET VS. NON-TARGET INVIRONMENTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Simultaneous improvement of forage traits and seed yield in orchardgrass (Dactylis glomerata L.) has been problematic because of geographic separation of forage and seed production locations. Previous work has shown that a complex multi-location selection program in forage production environments c...

  10. Antibody Drug Conjugates: Application of Quantitative Pharmacology in Modality Design and Target Selection.

    PubMed

    Sadekar, S; Figueroa, I; Tabrizi, M

    2015-07-01

    Antibody drug conjugates (ADCs) are a multi-component modality comprising of an antibody targeting a cell-specific antigen, a potent drug/payload, and a linker that can be processed within cellular compartments to release payload upon internalization. Numerous ADCs are being evaluated in both research and clinical settings within the academic and pharmaceutical industry due to their ability to selectively deliver potent payloads. Hence, there is a clear need to incorporate quantitative approaches during early stages of drug development for effective modality design and target selection. In this review, we describe a quantitative approach and framework for evaluation of the interplay between drug- and systems-dependent properties (i.e., target expression, density, localization, turnover, and affinity) in order to deliver a sufficient amount of a potent payload into the relevant target cells. As discussed, theoretical approaches with particular considerations given to various key properties for the target and modality suggest that delivery of the payload into particular effect cells to be more sensitive to antigen concentrations for targets with slow turnover rates as compared to those with faster internalization rates. Further assessments also suggest that increasing doses beyond the threshold of the target capacity (a function of target internalization and expression) may not impact the maximum amount of payload delivered to the intended effect cells. This article will explore the important application of quantitative sciences in selection of the target and design of ADC modalities. PMID:25933599

  11. Neural correlates of target selection for reaching movements in superior colliculus.

    PubMed

    Song, Joo-Hyun; McPeek, Robert M

    2015-03-01

    We recently demonstrated that inactivation of the primate superior colliculus (SC) causes a deficit in target selection for arm-reaching movements when the reach target is located in the inactivated field (Song JH, Rafal RD, McPeek RM. Proc Natl Acad Sci USA 108: E1433-E1440, 2011). This is consistent with the notion that the SC is part of a general-purpose target selection network beyond eye movements. To understand better the role of SC activity in reach target selection, we examined how individual SC neurons in the intermediate layers discriminate a reach target from distractors. Monkeys reached to touch a color oddball target among distractors while maintaining fixation. We found that many SC neurons robustly discriminate the goal of the reaching movement before the onset of the reach even though no saccade is made. To identify these cells in the context of conventional SC cell classification schemes, we also recorded visual, delay-period, and saccade-related responses in a delayed saccade task. On average, SC cells that discriminated the reach target from distractors showed significantly higher visual and delay-period activity than nondiscriminating cells, but there was no significant difference in saccade-related activity. Whereas a majority of SC neurons that discriminated the reach target showed significant delay-period activity, all nondiscriminating cells lacked such activity. We also found that some cells without delay-period activity did discriminate the reach target from distractors. We conclude that the majority of intermediate-layer SC cells discriminate a reach target from distractors, consistent with the idea that the SC contains a priority map used for effector-independent target selection. PMID:25505107

  12. Concept for On-Board Safe Landing Target Selection and Landing for the Mars 2020 Mission

    NASA Astrophysics Data System (ADS)

    Brugarolas, P.; Chen, A.; Johnson, A.; Casoliva, J.; Singh, G.; Stehura, A.; Way, D.; Dutta, S.

    2014-06-01

    We present a concept for a potential enhancement to Mars 2020 to enable landing on hazardous landing sites. It adds to MSL-EDL the capability to select and divert to a safe site through on-board terrain relative localization and target selection.

  13. Quantifying the tendency of therapeutic target proteins to bind promiscuous or selective compounds.

    PubMed

    Hu, Ye; Bajorath, Jrgen

    2015-01-01

    The ability of target proteins to bind structurally diverse compounds and compounds with different degrees of promiscuity (multi-target activity) was systematically assessed on the basis of currently available activity data and target annotations. Intuitive first- and second-order target promiscuity indices were introduced to quantify these binding characteristics and relate them to each other. For compounds and targets, opposite promiscuity trends were observed. Furthermore, the analysis detected many targets that interacted with compounds representing a similar degree of structural diversity but displayed strong tendencies to recognize either promiscuous or selective compounds. Moreover, target families were identified that preferentially interacted with promiscuous compounds. Taken together, these findings further extend our understanding of the molecular basis of polypharmacology. PMID:26000736

  14. The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.

    PubMed

    Sleat, David E; Lackland, Henry; Wang, Yanhong; Sohar, Istvan; Xiao, Gang; Li, Hong; Lobel, Peter

    2005-04-01

    The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes. PMID:15789345

  15. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes.

    PubMed

    Zeng, Jibin; Racicott, Jesse; Morales, Carlos R

    2009-11-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM(2)AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice. PMID:19732768

  16. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

    SciTech Connect

    Zeng, Jibin; Racicott, Jesse; Morales, Carlos R.

    2009-11-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM{sub 2}AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.

  17. Chemical screen to reduce sterol accumulation in Niemann-Pick C disease cells identifies novel lysosomal acid lipase inhibitors.

    PubMed

    Rosenbaum, Anton I; Rujoi, Madalina; Huang, Amy Y; Du, Hong; Grabowski, Gregory A; Maxfield, Frederick R

    2009-12-01

    Niemann-Pick C disease (NPC) is a lysosomal storage disorder causing abnormal accumulation of unesterified free cholesterol in lysosomal storage organelles. High content phenotypic microscopy chemical screens in both human and hamster NPC-deficient cells have identified several compounds that partially revert the NPC phenotype. Cell biological and biochemical studies show that several of these molecules inhibit lysosomal acid lipase, the enzyme that hydrolyzes LDL-derived triacylglycerol and cholesteryl esters. The effects of reduced lysosomal acid lipase activity in lowering cholesterol accumulation in NPC mutant cells were verified by RNAi-mediated knockdown of lysosomal acid lipase in NPC1-deficient human fibroblasts. This work demonstrates the utility of phenotypic cellular screens as a means to identify molecular targets for altering a complex process such as intracellular cholesterol trafficking and metabolism. PMID:19699313

  18. Engineering of Targeted Nanoparticles for Cancer Therapy Using Internalizing Aptamers Isolated by Cell-Uptake Selection

    PubMed Central

    Xiao, Zeyu; Levy-Nissenbaum, Etgar; Alexis, Frank; Lupták, Andrej; Teply, Benjamin A.; Chan, Juliana M.; Shi, Jinjun; Digga, Elise; Cheng, Judy; Langer, Robert; Farokhzad, Omid C.

    2012-01-01

    One of the major challenges in the development of targeted nanoparticles (NPs) for cancer therapy is to discover targeting ligands that allow for differential binding and uptake by the target cancer cells. Using prostate cancer (PCa) as a model disease, we developed a cell-uptake selection strategy to isolate PCa-specific internalizing 2'-Omethyl RNA aptamers (Apts) for NP incorporation. Twelve cycles of selection and counter-selection were done to obtain a panel of internalizing Apts, which can distinguish PCa cells from non-prostate and normal prostate cells. After Apt characterization, size minimization, and conjugation of the Apts with fluorescently-labeled polymeric NPs, the NP-Apt bioconjugates exhibit PCa specificity and enhancement in cellular uptake when compared to non-targeted NPs lacking the internalizing Apts. Furthermore, when docetaxel, a chemotherapeutic agent used for the treatment of PCa, was encapsulated within the NP-Apt, a significant improvement in cytotoxicity was achieved in targeted PCa cells. Rather than isolating high-affinity Apts as reported in previous selection processes, our selection strategy was designed to enrich cancer-cell specific internalizing Apts. A similar cell-uptake selection strategy may be used to develop specific internalizing ligands for a myriad of other diseases and can potentially facilitate delivering various molecules, including drugs and siRNAs, into cells. PMID:22214176

  19. Targeting Protein Kinases with Selective and Semi-Promiscuous Covalent Inhibitors

    PubMed Central

    Miller, Rand M.; Taunton, Jack

    2014-01-01

    Protein kinase inhibitors are an important class of therapeutics. In addition, selective kinase inhibitors can often reveal unexpected biological insights, augmenting genetic approaches and playing a decisive role in preclinical target validation studies. Nevertheless, developing protein kinase inhibitors with sufficient selectivity and pharmacodynamic potency presents significant challenges. Targeting noncatalytic cysteines with covalent inhibitors is a powerful approach to address both challenges simultaneously. Here, we describe our efforts to design irreversible and reversible electrophilic inhibitors with varying degrees of kinase selectivity. Highly selective covalent inhibitors have been used to elucidate the roles of p90 ribosomal protein S6 kinases (RSK) in animal models of atherosclerosis and diabetes. By contrast, semi-promiscuous covalent inhibitors have revealed new therapeutic targets in disease-causing parasites and have shown utility as chemoproteomic probes for interrogating kinase occupancy in living cells. PMID:25399643

  20. PPAR? in lysosomal biogenesis: A perspective.

    PubMed

    Ghosh, Arunava; Pahan, Kalipada

    2016-01-01

    Lysosomes are membrane-bound vesicles containing hydrolytic enzymes, ubiquitously present in all eukaryotic cells. Classically considered to be central to the cellular waste management machinery, recent studies revealed the role of lysosomes in a wide array of cellular processes like, degradation, cellular development, programmed cell death, secretion, plasma membrane repair, nutritional responses, and lipid metabolism. We recently studied the regulation of TFEB, considered to be the master regulator of lysosomal biogenesis, by activation of peroxisomal proliferator activated receptor ? (PPAR?), one of the key regulators of lipid metabolism. In this article, we discuss how the recent finding could be put in to perspective with the previous findings that relate lysosomal biogenesis to lipid metabolism, and comment on the possibility of a bi-directional interplay between these two distinct cellular processes upon activation of PPAR?. PMID:26621249

  1. Lysosomal proteins in cell death and autophagy.

    PubMed

    Mrschtik, Michaela; Ryan, Kevin M

    2015-05-01

    Nearly 60 years ago, lysosomes were first described in the laboratory of Christian de Duve, a discovery that significantly contributed to him being awarded a share of the 1974 Nobel Prize in Physiology or Medicine for elucidating 'the structural and functional organization of the cell'. Initially thought of as a simple waste degradation facility of the cell, these organelles recently emerged as signalling centres with connections to major cellular processes. This review provides an overview of the many roles of lysosomal proteins in two of these processes: cell death and autophagy. We discuss both resident lysosomal proteins as well those that temporarily associate with lysosomes to influence autophagy and cell death pathways. Particular focus is given to studies in mammalian cells and in vivo systems. PMID:25735653

  2. Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

    PubMed

    Rega, Laura R; Polishchuk, Elena; Montefusco, Sandro; Napolitano, Gennaro; Tozzi, Giulia; Zhang, Jinzhong; Bellomo, Francesco; Taranta, Anna; Pastore, Anna; Polishchuk, Roman; Piemonte, Fiorella; Medina, Diego L; Catz, Sergio D; Ballabio, Andrea; Emma, Francesco

    2016-04-01

    Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis. PMID:26994576

  3. Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

    PubMed Central

    Polishchuk, ElenaV.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; vanIJzendoorn, SvenC.D.; Chan, Jefferson; Chang, ChristopherJ.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, RomanS.

    2014-01-01

    Summary Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease. PMID:24909901

  4. The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization

    PubMed Central

    Stamelos, Vasileios A.; Fisher, Natalie; Bamrah, Harnoor; Voisey, Carolyn; Price, Joshua C.; Farrell, William E.; Redman, Charles W.; Richardson, Alan

    2016-01-01

    Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal alkalinization contributes to the cytotoxic activity of obatoclax. PMID:26950068

  5. Lysosomal storage diseases and the heat shock response: convergences and therapeutic opportunities

    PubMed Central

    Ingemann, Linda; Kirkegaard, Thomas

    2014-01-01

    Lysosomes play a vital role in the maintenance of cellular homeostasis through the recycling of cell constituents, a key metabolic function which is highly dependent on the correct function of the lysosomal hydrolases and membrane proteins, as well as correct membrane lipid stoichiometry and composition. The critical role of lysosomal functionality is evident from the severity of the diseases in which the primary lesion is a genetically defined loss-of-function of lysosomal hydrolases or membrane proteins. This group of diseases, known as lysosomal storage diseases (LSDs), number more than 50 and are associated with severe neurodegeneration, systemic disease, and early death, with only a handful of the diseases having a therapeutic option. Another key homeostatic system is the metabolic stress response or heat shock response (HSR), which is induced in response to a number of physiological and pathological stresses, such as protein misfolding and aggregation, endoplasmic reticulum stress, oxidative stress, nutrient deprivation, elevated temperature, viral infections, and various acute traumas. Importantly, the HSR and its cardinal members of the heat shock protein 70 family has been shown to protect against a number of degenerative diseases, including severe diseases of the nervous system. The cytoprotective actions of the HSR also include processes involving the lysosomal system, such as cell death, autophagy, and protection against lysosomal membrane permeabilization, and have shown promise in a number of LSDs. This review seeks to describe the emerging understanding of the interplay between these two essential metabolic systems, the lysosomes and the HSR, with a particular focus on their potential as a therapeutic target for LSDs. PMID:24837749

  6. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the G? subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. PMID:26667172

  7. An integrative role for the superior colliculus in selecting targets for movements.

    PubMed

    Wolf, Andrew B; Lintz, Mario J; Costabile, Jamie D; Thompson, John A; Stubblefield, Elizabeth A; Felsen, Gidon

    2015-10-01

    A fundamental goal of systems neuroscience is to understand the neural mechanisms underlying decision making. The midbrain superior colliculus (SC) is known to be central to the selection of one among many potential spatial targets for movements, which represents an important form of decision making that is tractable to rigorous experimental investigation. In this review, we first discuss data from mammalian models-including primates, cats, and rodents-that inform our understanding of how neural activity in the SC underlies the selection of targets for movements. We then examine the anatomy and physiology of inputs to the SC from three key regions that are themselves implicated in motor decisions-the basal ganglia, parabrachial region, and neocortex-and discuss how they may influence SC activity related to target selection. Finally, we discuss the potential for methodological advances to further our understanding of the neural bases of target selection. Our overarching goal is to synthesize what is known about how the SC and its inputs act together to mediate the selection of targets for movements, to highlight open questions about this process, and to spur future studies addressing these questions. PMID:26203103

  8. FOXP2 targets show evidence of positive selection in European populations.

    PubMed

    Ayub, Qasim; Yngvadottir, Bryndis; Chen, Yuan; Xue, Yali; Hu, Min; Vernes, Sonja C; Fisher, Simon E; Tyler-Smith, Chris

    2013-05-01

    Forkhead box P2 (FOXP2) is a highly conserved transcription factor that has been implicated in human speech and language disorders and plays important roles in the plasticity of the developing brain. The pattern of nucleotide polymorphisms in FOXP2 in modern populations suggests that it has been the target of positive (Darwinian) selection during recent human evolution. In our study, we searched for evidence of selection that might have followed FOXP2 adaptations in modern humans. We examined whether or not putative FOXP2 targets identified by chromatin-immunoprecipitation genomic screening show evidence of positive selection. We developed an algorithm that, for any given gene list, systematically generates matched lists of control genes from the Ensembl database, collates summary statistics for three frequency-spectrum-based neutrality tests from the low-coverage resequencing data of the 1000 Genomes Project, and determines whether these statistics are significantly different between the given gene targets and the set of controls. Overall, there was strong evidence of selection of FOXP2 targets in Europeans, but not in the Han Chinese, Japanese, or Yoruba populations. Significant outliers included several genes linked to cellular movement, reproduction, development, and immune cell trafficking, and 13 of these constituted a significant network associated with cardiac arteriopathy. Strong signals of selection were observed for CNTNAP2 and RBFOX1, key neurally expressed genes that have been consistently identified as direct FOXP2 targets in multiple studies and that have themselves been associated with neurodevelopmental disorders involving language dysfunction. PMID:23602712

  9. FOXP2 Targets Show Evidence of Positive Selection in European Populations

    PubMed Central

    Ayub, Qasim; Yngvadottir, Bryndis; Chen, Yuan; Xue, Yali; Hu, Min; Vernes, Sonja C.; Fisher, Simon E.; Tyler-Smith, Chris

    2013-01-01

    Forkhead box P2 (FOXP2) is a highly conserved transcription factor that has been implicated in human speech and language disorders and plays important roles in the plasticity of the developing brain. The pattern of nucleotide polymorphisms in FOXP2 in modern populations suggests that it has been the target of positive (Darwinian) selection during recent human evolution. In our study, we searched for evidence of selection that might have followed FOXP2 adaptations in modern humans. We examined whether or not putative FOXP2 targets identified by chromatin-immunoprecipitation genomic screening show evidence of positive selection. We developed an algorithm that, for any given gene list, systematically generates matched lists of control genes from the Ensembl database, collates summary statistics for three frequency-spectrum-based neutrality tests from the low-coverage resequencing data of the 1000 Genomes Project, and determines whether these statistics are significantly different between the given gene targets and the set of controls. Overall, there was strong evidence of selection of FOXP2 targets in Europeans, but not in the Han Chinese, Japanese, or Yoruba populations. Significant outliers included several genes linked to cellular movement, reproduction, development, and immune cell trafficking, and 13 of these constituted a significant network associated with cardiac arteriopathy. Strong signals of selection were observed for CNTNAP2 and RBFOX1, key neurally expressed genes that have been consistently identified as direct FOXP2 targets in multiple studies and that have themselves been associated with neurodevelopmental disorders involving language dysfunction. PMID:23602712

  10. PLEKHM1: Adapting to life at the lysosome.

    PubMed

    McEwan, David G; Dikic, Ivan

    2015-04-01

    The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome. PMID:25905573

  11. Direct observation of lipoprotein cholesterol ester degradation in lysosomes.

    PubMed Central

    Lusa, S; Tanhuanpää, K; Ezra, T; Somerharju, P

    1998-01-01

    We have investigated whether pyrene-labelled cholesterol esters (PyrnCEs) (n indicates the number of aliphatic carbons in the pyrene-chain) can be used to observe the degradation of low-density lipoprotein (LDL)-derived cholesterol esters (CEs) in the lysosomes of living cells. To select the optimal substrates, hydrolysis of the PyrnCE species by lysosomal acid lipase (LAL) in detergent/phospholipid micelles was compared. The rate of hydrolysis varied markedly depending on the length of the pyrenyl chain. Pyr10CE was clearly the best substrate, while Pyr4CE was practically unhydrolysed. Pyr10CE and [3H]cholesteryl linoleate, the major CE species in LDL, were hydrolysed equally by LAL when incorporated together into reconstituted LDL (rLDL) particles, thus indicating that Pyr10CE is a reliable reporter of the lysosomal degradation of native CEs. When rLDL particles containing Pyr4CE or Pyr10CE were incubated with fibroblasts, the accumulation of bright intracellular vesicular fluorescence was observed with the former fluorescent derivative, but not with the latter. However, when the cells were treated with chloroquine, an inhibitor of lysosomal hydrolysis, or when cells with defective LAL were employed, Pyr10CE also accumulated in vesicular structures. HPLC analysis of cellular lipid extracts fully supported these imaging results. It is concluded that PyrnCEs can be used to observe degradation of CEs directly in living cells. This should be particularly useful when exploring the mechanisms responsible for the accumulation of lipoprotein-derived CEs in complex systems such as the arterial intima. PMID:9601074

  12. Changes during fasting in the activity of a specific lysosomal proteinase, fructose-1,6-bisphosphatase converting enzyme.

    PubMed Central

    Melloni, E; Pontremoli, S; Salamino, F; Sparatore, B; Michetti, M; Horecker, B L

    1981-01-01

    Three lysosomal proteinases capable of catalyzing a limited modification of the gluconeogenic enzyme fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) have been purified from extracts of rabbit liver lysosomes. These have been designated "converting enzymes I, II, and III," respectively, based on the order of their elution from Ultrogel AcA34. The predominant form in lysosomes from livers of fed rabbits is converting enzyme III which has been identified as a mixture of cathepsins B and L. Fasting induces a 4- to 5-fold increase in the activity of converting enzyme II; in the livers of 96-hr-fasted rabbits, this form accounts for more than 70% of the total converting enzyme activity. The increase is largely in that fraction of converting enzyme II associated with lysosomal membranes; this increase is also seen in the activity expressed by intact lysosomes. The activity of intact lysosomes is not due to their increased fragility because other lysosomal enzyme activities remain latent. The effect of fasting on the activity of converting enzyme II is selective and greatly exceeds the 2-fold increase observed for other lysosomal enzymes. PMID:6262806

  13. Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability

    PubMed Central

    Liu, Zhe; Liang, Chenyuan; Zhang, Zhuoyuan; Pan, Jian; Xia, Hui; Zhong, Nanshan

    2015-01-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells’ viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy. PMID:26302210

  14. Involvement of BimL activation in apoptosis induced by lysosomal photodamage

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Wang, Xianwang; Li, Hui

    2008-12-01

    Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using real-time single-cell analysis. Confocal imaging of cells transfected with BimL-GFP demonstrated that BimL translocated to mitochondria after NPe6-PDT treatment for about 150 min, and then sequestered into clusters associated with the mitochondira within 30 min. The activation of BimL proved to be an important event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples suppressed the expression level of endogenous BimL. This study demonstrates that BimL activation was involved in the cell death induced by PDT with lysosomal photosensitizer.

  15. Optimal Intermittence in Search Strategies under Speed-Selective Target Detection

    NASA Astrophysics Data System (ADS)

    Campos, Daniel; Mndez, Vicen; Bartumeus, Frederic

    2012-01-01

    Random search theory has been previously explored for both continuous and intermittent scanning modes with full target detection capacity. Here we present a new class of random search problems in which a single searcher performs flights of random velocities, the detection probability when it passes over a target location being conditioned to the searcher speed. As a result, target detection involves an N-passage process for which the mean search time is here analytically obtained through a renewal approximation. We apply the idea of speed-selective detection to random animal foraging since a fast movement is known to significantly degrade perception abilities in many animals. We show that speed-selective detection naturally introduces an optimal level of behavioral intermittence in order to solve the compromise between fast relocations and target detection capability.

  16. Comparison of the Cancer Gene Targeting and Biochemical Selectivities of All Targeted Kinase Inhibitors Approved for Clinical Use

    PubMed Central

    Uitdehaag, Joost C. M.; de Roos, Jeroen A. D. M.; van Doornmalen, Antoon M.; Prinsen, Martine B. W.; de Man, Jos; Tanizawa, Yoshinori; Kawase, Yusuke; Yoshino, Kohichiro; Buijsman, Rogier C.; Zaman, Guido J. R.

    2014-01-01

    The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013), and for six of these drugs, the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers, suggesting that cancers dependent on mutant CTNNB1 will respond to trametinib and other MEK inhibitors, and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR, ABL1 and BRAF(V600E)-driven cell growth, and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors, we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action. PMID:24651269

  17. Contextual control over selective attention: evidence from a two-target method.

    PubMed

    MacLellan, Ellen; Shore, David I; Milliken, Bruce

    2015-07-01

    Selective attention is generally studied with conflict tasks, using response time as the dependent measure. Here, we study the impact of selective attention to a first target, T1, presented simultaneously with a distractor, on the accuracy of subsequent encoding of a second target item, T2. This procedure produces an "attentional blink" (AB) effect much like that reported in other studies, and allowed us to study the influence of context on cognitive control with a novel method. In particular, we examined whether preparation to attend selectively to T1 had an impact on the selective encoding of T1 that would translate to report of T2. Preparation to attend selectively was manipulated by varying whether difficult selective attention T1 trials were presented in the context of other difficult selective attention T1 trials. The results revealed strong context effects of this nature, with smaller AB effects when difficult selective attention T1 trials were embedded in a context with many, rather than few, other difficult selective attention T1 trials. Further, the results suggest that both the trial-to-trial local context and the block-wide global context modulate performance in this task. PMID:25030812

  18. The exploitation of differential endocytic pathways in normal and tumor cells in the selective targeting of nanoparticulate chemotherapeutic agents

    PubMed Central

    Sahay, Gaurav; Kim, Jong Oh; Kabanov, Alexander V; Bronich, Tatiana K

    2011-01-01

    Polymeric micelles with cross-linked ionic cores of poly(methacrylic acid) and nonionic shell of poly(ethylene oxide) (cl-micelles) are shown here to readily internalize in epithelial cancer cells but not in normal epithelial cells that form tight junctions (TJ). The internalization of such cl-micelles in the cancer cells proceeded mainly through caveolae-mediated endocytosis. In confluent normal epithelial cells this endocytosis route was absent at the apical side and the cl-micelles sequestered in TJ regions of the cell membrane without entering the cells for at least 24 hours. Disruption of the TJ by calcium deprivation resulted in redistribution of cl-micelles inside the cells. In cancer cells following initial cellular entry the cl-micelles bypassed the early endosomes and reached the lysosomes within 30 minutes. This allowed designing cl-micelles with cytotoxic drug, doxorubicin, linked via pH-sensitive hydrazone bonds, which were cleaved in the acidic environment of lysosomes resulting in accumulation of the drug in the nucleus after 5 hours. Such pH-sensitive cl-micelles displayed selective toxicity to cancer cells but were nontoxic to normal epithelial cells. In conclusion, we describe major difference in interactions of cl-micelles with cancer and normal cells that can lead to development of novel drug delivery system with reduced side effects and higher efficacy in cancer chemotherapy. PMID:19853293

  19. A systematic bioinformatics approach for selection of epitope-based vaccine targets

    PubMed Central

    Khan, Asif M.; Miotto, Olivo; Heiny, A.T.; Salmon, Jerome; Srinivasan, K.N.; Nascimento, Eduardo; Marques, Ernesto T.; Brusic, Vladimir; Tan, Tin Wee; August, J. Thomas

    2007-01-01

    Epitope-based vaccines provide a new strategy for prophylactic and therapeutic application of pathogen-specific immunity. A critical requirement of this strategy is the identification and selection of T-cell epitopes that act as vaccine targets. This study describes current methodologies for the selection process, with dengue virus as a model system. A combination of publicly available bioinformatics algorithms and computational tools are used to screen and select antigen sequences as potential T-cell epitopes of supertype HLA alleles. The selected sequences are tested for biological function by their activation of T-cells of HLA transgenic mice and of pathogen infected subjects. This approach provides an experimental basis for the design of pathogen specific, T-cell epitope-based vaccines that are targeted to majority of the genetic variants of the pathogen, and are effective for a broad range of differences in human leukocyte antigens among the global human population. PMID:17434154

  20. Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM).

    PubMed

    Adachi, Jun; Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

    Mass spectrometry-based phosphoproteomics has been rapidly spread based on the advancement of mass spectrometry and development of efficient enrichment techniques for phosphorylated proteins or peptides. Non-targeted approach has been employed in most of the studies for phosphoproteome analysis. However, targeted approach using selected/multiple reaction monitoring (SRM/MRM) is an indispensible technique used for the quantitation of known targets especially when we have many samples to quantitate phosphorylation events on proteins in biological or clinical samples. We herein describe the application of a large-scale phosphoproteome analysis and SRM-based quantitation for the systematic discovery and validation of biomarkers. PMID:26700043

  1. Signaling from lysosomes enhances mitochondria-mediated photodynamic therapy in cancer cells

    NASA Astrophysics Data System (ADS)

    Quiogue, Geraldine; Saggu, Shalini; Hung, Hsin-I.; Kenney, Malcolm E.; Oleinick, Nancy L.; Lemasters, John J.; Nieminen, Anna-Liisa

    2009-06-01

    In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Assessed by confocal microscopy, the photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. A Pc 4 derivative, Pc 181, accumulates into lysosomes. In comparison to Pc 4, Pc 181 was a more effective photosensitizer promoting killing cancer cells after PDT. The mode of cell death after Pc 181-PDT is predominantly apoptosis, and pancaspase and caspase-3 inhibitors prevent onset of the cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431cells after PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself did not induce toxicity but greatly enhanced Pc 4-PDT-induced cell killing. In comparison to Pc 4, less enhancement of cell killing by bafilomycin occurred after Pc 181-PDT at photosensitizer doses producing equivalent cell killing in the absence of bafilomycin. These results indicate that lysosomal disruption can augment PDT with Pc 4, which targets predominantly mitochondria, but less so after PDT with Pc 181, since Pc 181 already targets lysosomes.

  2. [Medicinal therapy for lysosomal storage diseases].

    PubMed

    Sakuragawa, N

    1995-12-01

    Lysosomes contain several dozen different enzymes, mostly acid hydrolases. Materials not digested due to deficient lysosomal enzymes are usually large cellular molecules, which are deposited within the cells. The strategy for medicinal therapy of lysosomal storages disease may be to develop the activators of enzymes, to promote coenzyme and cofactor supplementation and to eliminate undegraded materials from blood into urine. In the last several decades, many trials for these strategies has been done. Cysteamine for cystinosis and penicillamine for Wilson's disease has proved useful in treating these patients. Recently, DMSO has been proved to be an activator of acid sphingomyelinase and to accelerate the intracellular mobilization of LDL-derived cholesterol. We treated patients with Niemann-Pick disease type C by oral administration of DMSO, resulting in some clinical benefits such as decreased size of hepatosplenomegaly, and lesser frequency of seizures with improved EEG. However, the progressive clinical course has not been changed although it appeared to slow down. New activators of lysosomal enzymes should be developed for medicinal therapy of lysosomal storage diseases. PMID:8577061

  3. Joint Effect of Insertion of Spaces and Word Length in Saccade Target Selection in Chinese Reading

    ERIC Educational Resources Information Center

    Li, Xingshan; Shen, Wei

    2013-01-01

    The present study examined how insertion of spaces before and after a word affects saccade target selection in Chinese reading. We found that inserting spaces in Chinese text changes the eye movement behaviour of Chinese readers. They are less likely to fixate on the character near the space and will try their best to process the entire word with…

  4. Competition between color and luminance for target selection in smooth pursuit and saccadic eye movements.

    PubMed

    Spering, Miriam; Montagnini, Anna; Gegenfurtner, Karl R

    2008-01-01

    Visual processing of color and luminance for smooth pursuit and saccadic eye movements was investigated using a target selection paradigm. In two experiments, stimuli were varied along the dimensions color and luminance, and selection of the more salient target was compared in pursuit and saccades. Initial pursuit was biased in the direction of the luminance component whereas saccades showed a relative preference for color. An early pursuit response toward luminance was often reversed to color by a later saccade. Observers' perceptual judgments of stimulus salience, obtained in two control experiments, were clearly biased toward luminance. This choice bias in perceptual data implies that the initial short-latency pursuit response agrees with perceptual judgments. In contrast, saccades, which have a longer latency than pursuit, do not seem to follow the perceptual judgment of salience but instead show a stronger relative preference for color. These substantial differences in target selection imply that target selection processes for pursuit and saccadic eye movements use distinctly different weights for color and luminance stimuli. PMID:19146301

  5. Strategies for the Discovery of Target-Specific or Isoform-Selective Modulators.

    PubMed

    Zhan, Peng; Itoh, Yukihiro; Suzuki, Takayoshi; Liu, Xinyong

    2015-10-01

    Currently, the creation of class- and isoform-selective modulators of biologically important targets is a particularly challenging problem because different isoforms within a protein family often show striking similarity in spatial quaternary structure, especially at the catalytic sites or binding pockets. Therefore, an understanding of both the precise three-dimensional structure of the target protein and the mechanisms of action of modulators is important for developing more effective and selective agents. In this Perspective, we discuss currently available rational design strategies for obtaining class- and isoform-selective inhibitors and we illustrate these strategies with the aid of specific examples from the recent literature. The strategies covered include: (1) target-derived (-dependent) de novo drug discovery methodologies, and (2) follow-on derivatization approaches from initially identified active molecules (hit-to-lead and lead-to-candidate efforts). We also comment on prospects for further development and integration of strategies to achieve target-specific or isoform-selective inhibition. PMID:26086931

  6. Target Selection for the Arecibo Pisces-Perseus Supercluster Survey (APPSS)

    NASA Astrophysics Data System (ADS)

    Craig, David W.; O'Donoghue, Aileen A.; Haynes, Martha P.; Rosenberg, Jessica L.; Venkatesan, Aparna; Hallenbeck, Gregory L.; Jones, Michael; Koopmann, Rebecca A.; Undergraduate ALFALFA Team

    2016-01-01

    The Arecibo Pisces-Perseus Supercluster Survey (APPSS) is a new large targeted HI survey now underway using Arecibo's L-band Wide receiver system. A major goal is to constrain models of the Pisces Perseus infall, producing 5-σ detections of infall motions ˜500 km s-1. We are targeting sources that are likely to be at the PPS distance, but that are just below the the HI mass detection threshold of the ALFALFA survey. We expect to identify ˜800 objects of mass ˜108—9 M⊙ which will alllow us to constrain the lower mass end of the HI mass function in this infall environment.We have pursued a multi-pronged approach to target selection for this survey. Sources from ALFALFA, SDSS, and the GALEX GCAT single source catalogs were matched and intercompared via multi-band color photometry, surface brightnesses, and appearance in SDSS images. Final target selection based on visual inspection of SDSS images was found to correlate well with a color-selection technique based on GALEX/NUV - SDSS/r. Along with the details of the source selection we will discuss the facilitation and implementation of this process via a multi-institution collaborative website, and early results from the APSS survey.This work has been supported by NSF grant AST-1211005.

  7. TARGET SELECTION FOR THE APACHE POINT OBSERVATORY GALACTIC EVOLUTION EXPERIMENT (APOGEE)

    SciTech Connect

    Zasowski, G.; Johnson, Jennifer A.; Andrews, B.; Epstein, C.; Frinchaboy, P. M.; Jackson, K.; Majewski, S. R.; Chojnowski, S. D.; Skrutskie, M. F.; Beaton, R. L.; Nidever, D. L.; Pinto, H. J. Rocha; Girardi, L.; Cudworth, K. M.; Munn, J.; Blake, C. H.; Covey, K.; Deshpande, R.; Fleming, S. W.; Fabbian, D. [Instituto de Astrofisica de Canarias, Calle Via Lactea s and others

    2013-10-01

    The Apache Point Observatory Galactic Evolution Experiment (APOGEE) is a high-resolution infrared spectroscopic survey spanning all Galactic environments (i.e., bulge, disk, and halo), with the principal goal of constraining dynamical and chemical evolution models of the Milky Way. APOGEE takes advantage of the reduced effects of extinction at infrared wavelengths to observe the inner Galaxy and bulge at an unprecedented level of detail. The survey's broad spatial and wavelength coverage enables users of APOGEE data to address numerous Galactic structure and stellar populations issues. In this paper we describe the APOGEE targeting scheme and document its various target classes to provide the necessary background and reference information to analyze samples of APOGEE data with awareness of the imposed selection criteria and resulting sample properties. APOGEE's primary sample consists of {approx}10{sup 5} red giant stars, selected to minimize observational biases in age and metallicity. We present the methodology and considerations that drive the selection of this sample and evaluate the accuracy, efficiency, and caveats of the selection and sampling algorithms. We also describe additional target classes that contribute to the APOGEE sample, including numerous ancillary science programs, and we outline the targeting data that will be included in the public data releases.

  8. Target-selective Protein S-Nitrosylation by Sequence Motif Recognition

    PubMed Central

    Jia, Jie; Arif, Abul; Terenzi, Fulvia; Willard, Belinda; Plow, Edward F.; Hazen, Stanley L.; Fox, Paul L.

    2014-01-01

    SUMMARY S-nitrosylation is a ubiquitous protein modification emerging as a principal mechanism of nitric oxide (NO)-mediated signal transduction and cell function. S-nitrosylases can use NO synthase (NOS)-derived NO to modify selected cysteines in target proteins. Despite proteomic identification of more than a thousand S-nitrosylated proteins, very few S-nitrosylases have been identified. Moreover, mechanisms underlying site-selective S-nitrosylation and the potential role of specific sequence motifs remain largely unknown. Here, we describe a stimulus-inducible, heterotrimeric S-nitrosylase complex consisting of inducible NOS (iNOS), S100A8, and S100A9. S100A9 exhibits transnitrosylase activity, shuttling NO from iNOS to the target protein, whereas S100A8 and S100A9 coordinately direct site selection. A family of proteins S-nitrosylated by the iNOS-S100A8/A9 complex were revealed by proteomic analysis, and validated as targets. A conserved I/L-X-C-X2-D/E motif was necessary and sufficient for iNOS- S100A8/A9-mediated S-nitrosylation. These results reveal an elusive parallel between protein S-nitrosylation and phosphorylation, namely, stimulus-dependent post-translational modification of selected targets by primary sequence motif recognition. PMID:25417112

  9. Joint Effect of Insertion of Spaces and Word Length in Saccade Target Selection in Chinese Reading

    ERIC Educational Resources Information Center

    Li, Xingshan; Shen, Wei

    2013-01-01

    The present study examined how insertion of spaces before and after a word affects saccade target selection in Chinese reading. We found that inserting spaces in Chinese text changes the eye movement behaviour of Chinese readers. They are less likely to fixate on the character near the space and will try their best to process the entire word with

  10. Efficient conditional knockout targeting vector construction using co-selection BAC recombineering (CoSBR)

    PubMed Central

    Newman, Robert J.; Roose-Girma, Merone; Warming, Sren

    2015-01-01

    A simple and efficient strategy for Bacterial Artificial Chromosome (BAC) recombineering based on co-selection is described. We show that it is possible to efficiently modify two positions of a BAC simultaneously by co-transformation of a single-stranded DNA oligo and a double-stranded selection cassette. The use of co-selection BAC recombineering reduces the DNA manipulation needed to make a conditional knockout gene targeting vector to only two steps: a single round of BAC modification followed by a retrieval step. PMID:26089387

  11. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    PubMed Central

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  12. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors.

    PubMed

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R J

    2015-11-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  13. Sequence-selective targeting of duplex DNA by peptide nucleic acids.

    PubMed

    Nielsen, Peter E

    2010-04-01

    Sequence-selective gene targeting constitutes an attractive drug-discovery approach for genetic therapy, with the aim of reducing or enhancing the activity of specific genes at the transcriptional level, or as part of a methodology for targeted gene repair. The pseudopeptide DNA mimic peptide nucleic acid (PNA) can recognize duplex DNA with high sequence specificity and affinity in triplex, duplex and double-duplex invasive modes or non-invasive triplex modes. Novel PNA modification has improved the affinity for DNA recognition via duplex invasion, double-duplex invasion and triplex recognition considerably. Such modifications have also resulted in new approaches to targeted gene repair and sequence-selective double-strand cleavage of genomic DNA. PMID:20373262

  14. Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells.

    PubMed

    Sachlos, Eleftherios; Risueo, Ruth M; Laronde, Sarah; Shapovalova, Zoya; Lee, Jong-Hee; Russell, Jennifer; Malig, Monika; McNicol, Jamie D; Fiebig-Comyn, Aline; Graham, Monica; Levadoux-Martin, Marilyne; Lee, Jung Bok; Giacomelli, Andrew O; Hassell, John A; Fischer-Russell, Daniela; Trus, Michael R; Foley, Ronan; Leber, Brian; Xenocostas, Anargyros; Brown, Eric D; Collins, Tony J; Bhatia, Mickie

    2012-06-01

    Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy. PMID:22632761

  15. Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes*

    PubMed Central

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A.

    2010-01-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca2+ selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels. PMID:20495006

  16. Autonomous target dependent waveband selection for tracking in performance-driven hyperspectral sensing

    NASA Astrophysics Data System (ADS)

    Gadaleta, Sabino M.; Kerekes, John P.; Tarplee, Kyle M.

    2012-06-01

    Performance-driven sensing is a promising new concept that relies on sensing, processing, and exploiting only the most "decision-relevant" sets of target data for the purpose of reducing requirements on data collection, processing, and communications. An example of a device supporting such a concept is a MEMS-based single pixel Fabry-Perot spectrometer being developed at the Rochester Institute of Technology, which can record selected wavelengths on a per-pixel basis throughout an image. This paper presents an autonomous target-dependent waveband selection approach for performance-driven sensing with an adaptive hyperspectral imaging sensor. Given a target that is to be tracked, a subset of wavebands is estimated from locally recorded hyperspectral data that provides optimal target detectability against local background. The waveband selection algorithm relies on finding a subset of bands that provides maximum separation between a target histogram and local background histogram constructed from the respective bands. To illustrate the concept, we perform a simulation study for vehicle tracking in a set of synthetic DIRSIG rendered HSI images. The simulations demonstrate improved vehicle tracking accuracy when using the adaptively-selected subset of wavebands for tracking by histogram matching compared to performing tracking by histogram matching with regular (fixed) color bands. We extend the framework to a dynamic concept where the waveband subset is updated over time as a function of position estimation accuracy and discuss the full integration of the Feature-Aided Tracking (FAT) component derived from the selected wavebands within a Multiple Hypothesis Tracking (MHT) framework.

  17. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

    PubMed Central

    Ojo, Kayode K; Larson, Eric T; Keyloun, Katelyn R; Castaneda, Lisa J; DeRocher, Amy E; Inampudi, Krishna K; Kim, Jessica E; Arakaki, Tracy L; Murphy, Ryan C; Zhang, Li; Napuli, Alberto J; Maly, Dustin J; Verlinde, Christophe LMJ; Buckner, Frederick S; Parsons, Marilyn; Hol, Wim GJ; Merritt, Ethan A; Van Voorhis, Wesley C

    2010-01-01

    New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable. PMID:20436472

  18. Arylsulfatase K, a novel lysosomal sulfatase.

    PubMed

    Wiegmann, Elena Marie; Westendorf, Eva; Kalus, Ina; Pringle, Thomas H; Lbke, Torben; Dierks, Thomas

    2013-10-18

    The human sulfatase family has 17 members, 13 of which have been characterized biochemically. These enzymes specifically hydrolyze sulfate esters in glycosaminoglycans, sulfolipids, or steroid sulfates, thereby playing key roles in cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked to severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase K (ARSK), was identified bioinformatically through its conserved sulfatase signature sequence directing posttranslational generation of the catalytic formylglycine residue in sulfatases. However, overall sequence identity of ARSK with other human sulfatases is low (18-22%). Here we demonstrate that ARSK indeed shows desulfation activity toward arylsulfate pseudosubstrates. When expressed in human cells, ARSK was detected as a 68-kDa glycoprotein carrying at least four N-glycans of both the complex and high-mannose type. Purified ARSK turned over p-nitrocatechol and p-nitrophenyl sulfate. This activity was dependent on cysteine 80, which was verified to undergo conversion to formylglycine. Kinetic parameters were similar to those of several lysosomal sulfatases involved in degradation of sulfated glycosaminoglycans. An acidic pH optimum (~4.6) and colocalization with LAMP1 verified lysosomal functioning of ARSK. Further, it carries mannose 6-phosphate, indicating lysosomal sorting via mannose 6-phosphate receptors. ARSK mRNA expression was found in all tissues tested, suggesting a ubiquitous physiological substrate and a so far non-classified lysosomal storage disorder in the case of ARSK deficiency, as shown before for all other lysosomal sulfatases. PMID:23986440

  19. Arylsulfatase K, a Novel Lysosomal Sulfatase*

    PubMed Central

    Wiegmann, Elena Marie; Westendorf, Eva; Kalus, Ina; Pringle, Thomas H.; Lbke, Torben; Dierks, Thomas

    2013-01-01

    The human sulfatase family has 17 members, 13 of which have been characterized biochemically. These enzymes specifically hydrolyze sulfate esters in glycosaminoglycans, sulfolipids, or steroid sulfates, thereby playing key roles in cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked to severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase K (ARSK), was identified bioinformatically through its conserved sulfatase signature sequence directing posttranslational generation of the catalytic formylglycine residue in sulfatases. However, overall sequence identity of ARSK with other human sulfatases is low (1822%). Here we demonstrate that ARSK indeed shows desulfation activity toward arylsulfate pseudosubstrates. When expressed in human cells, ARSK was detected as a 68-kDa glycoprotein carrying at least four N-glycans of both the complex and high-mannose type. Purified ARSK turned over p-nitrocatechol and p-nitrophenyl sulfate. This activity was dependent on cysteine 80, which was verified to undergo conversion to formylglycine. Kinetic parameters were similar to those of several lysosomal sulfatases involved in degradation of sulfated glycosaminoglycans. An acidic pH optimum (?4.6) and colocalization with LAMP1 verified lysosomal functioning of ARSK. Further, it carries mannose 6-phosphate, indicating lysosomal sorting via mannose 6-phosphate receptors. ARSK mRNA expression was found in all tissues tested, suggesting a ubiquitous physiological substrate and a so far non-classified lysosomal storage disorder in the case of ARSK deficiency, as shown before for all other lysosomal sulfatases. PMID:23986440

  20. DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX

    PubMed Central

    Guan, J-J; Zhang, X-D; Sun, W; Qi, L; Wu, J-C; Qin, Z-H

    2015-01-01

    DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis. PMID:25633293

  1. Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin.

    PubMed

    Ivanova, Ekaterina A; De Leo, Maria Giovanna; Van Den Heuvel, Lambertus; Pastore, Anna; Dijkman, Henry; De Matteis, Maria Antonietta; Levtchenko, Elena N

    2015-01-01

    Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles. PMID:25811383

  2. Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal Cystine Transporter Cystinosin

    PubMed Central

    Van Den Heuvel, Lambertus; Pastore, Anna; Dijkman, Henry; De Matteis, Maria Antonietta; Levtchenko, Elena N.

    2015-01-01

    Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles. PMID:25811383

  3. Stimulus selection and tracking during urination: autoshaping directed behavior with toilet targets.

    PubMed Central

    Siegel, R K

    1977-01-01

    A simple procedure is described for investigating stimuli selected as targets during urination in the commode. Ten normal males preferred a floating target that could be tracked to a series of stationary targets. This technique was used to bring misdirected urinations in a severely retarded male under rapid stimulus control of a floating target in the commode. The float stimulus was also evaluated with nine institionalized, moderately retarded males and results indicated rapid autoshaping of directed urination without the use of verbal instructions or conventional toilet training. The technique can be applied in training children to control misdirected urinations in institution for the retarded, in psychiatric wards with regressed populations, and in certain male school dormitories. PMID:885828

  4. A fluorescent indicator for imaging lysosomal zinc(II) with Frster resonance energy transfer (FRET)-enhanced photostability and a narrow band of emission.

    PubMed

    Sreenath, Kesavapillai; Yuan, Zhao; Allen, John R; Davidson, Michael W; Zhu, Lei

    2015-01-01

    We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Frster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells. PMID:25382395

  5. A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Frster Resonance Energy Transfer (FRET)-Enhanced Photostability and a Narrow Band of Emission.

    PubMed

    Sreenath, Kesavapillai; Yuan, Zhao; Allen, John R; Davidson, Michael W; Zhu, Lei

    2014-11-01

    We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Frster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells. PMID:25378058

  6. A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)-Enhanced Photostability and a Narrow Band of Emission

    PubMed Central

    Sreenath, Kesavapillai; Yuan, Zhao; Allen, John R.

    2015-01-01

    We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells. PMID:25382395

  7. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

    PubMed

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

    2014-12-19

    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. PMID:25342746

  8. A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants

    PubMed Central

    Muehlbauer, Stefan M.; Chandran, Kartik; Diaz-Griffero, Felipe

    2014-01-01

    Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants. PMID:24893007

  9. A new lactoferrin- and iron-dependent lysosomal death pathway is induced by benzo[a]pyrene in hepatic epithelial cells

    SciTech Connect

    Gorria, Morgane; Tekpli, Xavier; Rissel, Mary |; Sergent, Odile; Huc, Laurence |; Landvik, Nina; Fardel, Olivier; Dimanche-Boitrel, Marie-Therese |; Holme, Jorn A.; Lagadic-Gossmann, Dominique |

    2008-04-15

    While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation and cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.

  10. Extreme selective sweeps independently targeted the X chromosomes of the great apes

    PubMed Central

    Nam, Kiwoong; Munch, Kasper; Hobolth, Asger; Dutheil, Julien Yann; Veeramah, Krishna R.; Woerner, August E.; Hammer, Michael F.; Mailund, Thomas; Schierup, Mikkel Heide

    2015-01-01

    The unique inheritance pattern of the X chromosome exposes it to natural selection in a way that is different from that of the autosomes, potentially resulting in accelerated evolution. We perform a comparative analysis of X chromosome polymorphism in 10 great ape species, including humans. In most species, we identify striking megabase-wide regions, where nucleotide diversity is less than 20% of the chromosomal average. Such regions are found exclusively on the X chromosome. The regions overlap partially among species, suggesting that the underlying targets are partly shared among species. The regions have higher proportions of singleton SNPs, higher levels of population differentiation, and a higher nonsynonymous-to-synonymous substitution ratio than the rest of the X chromosome. We show that the extent to which diversity is reduced is incompatible with direct selection or the action of background selection and soft selective sweeps alone, and therefore, we suggest that very strong selective sweeps have independently targeted these specific regions in several species. The only genomic feature that we can identify as strongly associated with loss of diversity is the location of testis-expressed ampliconic genes, which also have reduced diversity around them. We hypothesize that these genes may be responsible for selective sweeps in the form of meiotic drive caused by an intragenomic conflict in male meiosis. PMID:25941379

  11. Diverse selective regimes shape genetic diversity at ADAR genes and at their coding targets.

    PubMed

    Forni, Diego; Mozzi, Alessandra; Pontremoli, Chiara; Vertemara, Jacopo; Pozzoli, Uberto; Biasin, Mara; Bresolin, Nereo; Clerici, Mario; Cagliani, Rachele; Sironi, Manuela

    2015-01-01

    A-to-I RNA editing operated by ADAR enzymes is extremely common in mammals. Several editing events in coding regions have pivotal physiological roles and affect protein sequence (recoding events) or function. We analyzed the evolutionary history of the 3 ADAR family genes and of their coding targets. Evolutionary analysis indicated that ADAR evolved adaptively in primates, with the strongest selection in the unique N-terminal domain of the interferon-inducible isoform. Positively selected residues in the human lineage were also detected in the ADAR deaminase domain and in the RNA binding domains of ADARB1 and ADARB2. During the recent history of human populations distinct variants in the 3 genes increased in frequency as a result of local selective pressures. Most selected variants are located within regulatory regions and some are in linkage disequilibrium with eQTLs in monocytes. Finally, analysis of conservation scores of coding editing sites indicated that editing events are counter-selected within regions that are poorly tolerant to change. Nevertheless, a minority of recoding events occurs at highly conserved positions and possibly represents the functional fraction. These events are enriched in pathways related to HIV-1 infection and to epidermis/hair development. Thus, both ADAR genes and their targets evolved under variable selective regimes, including purifying and positive selection. Pressures related to immune response likely represented major drivers of evolution for ADAR genes. As for their coding targets, we suggest that most editing events are slightly deleterious, although a minority may be beneficial and contribute to antiviral response and skin homeostasis. PMID:25826567

  12. Glioma Selectivity of Magnetically Targeted Nanoparticles: A Role of Abnormal Tumor Hydrodynamics

    PubMed Central

    Chertok, Beata; David, Allan E.; Huang, Yongzhuo; Yang, Victor C.

    2007-01-01

    Magnetic targeting is a promising strategy for achieving localized drug delivery. Application of this strategy to treat brain tumors, however, is complicated by their deep intracranial location, since magnetic field density cannot be focused at a distance from an externally applied magnet. This study intended to examine whether, with magnetic targeting, pathological alteration in brain tumor flow dynamics could be of value in discriminating the diseased site from healthy brain. To address this question, the capture of magnetic nanoparticles was first assessed in vitro using a simple flow system under theoretically estimated glioma and normal brain flow conditions. Secondly, accumulation of nanoparticles via magnetic targeting was evaluated in vivo using 9L-glioma bearing rats. In vitro results that predicted a 7.6-fold increase in nanoparticle capture at glioma-versus contralateral brain-relevant flow rates were relatively consistent with the 9.6-fold glioma selectivity of nanoparticle accumulation over the contralateral brain observed in vivo. Based on these finding, the in vitro ratio of nanoparticle capture can be viewed as a plausible indicator of in vivo glioma selectivity. Overall, it can be concluded that the decreased blood flow rate in glioma, reflecting tumor vascular abnormalities, is an important contributor to glioma-selective nanoparticle accumulation with magnetic targeting. PMID:17628157

  13. Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents

    PubMed Central

    Modica-Napolitano, Josephine S.; Weissig, Volkmar

    2015-01-01

    Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including those that induce mitochondria permeability transition and apoptosis, metabolic inhibitors, and ROS regulators. To date, however, none has exhibited the standards for high selectivity and efficacy and low toxicity necessary to progress beyond phase III clinical trials and be used as a viable, single modality treatment option for human cancers. This review explores alternative treatment strategies that have been shown to enhance the efficacy and selectivity of mitochondria-targeted anticancer agents in vitro and in vivo, and may yet fulfill the clinical promise of exploiting the mitochondrion as a target for cancer chemotherapy. PMID:26230693

  14. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

    PubMed Central

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

  15. Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells

    PubMed Central

    Paliouras, Miltiadis; Mitmaker, Elliot J.; Trifiro, Mark A.

    2016-01-01

    Background The incidence of papillary thyroid carcinoma (PTC) has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid) act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches. Methods Thyroid Stimulating Hormone Receptor (TSHR) was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin) were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm) was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining. Results TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls. Conclusion A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam radiation or chemotherapy, with fewer side effects and improved quality of life. PMID:26901566

  16. Target selection and comparison of mission design for space debris removal by DLR's advanced study group

    NASA Astrophysics Data System (ADS)

    van der Pas, Niels; Lousada, Joao; Terhes, Claudia; Bernabeu, Marc; Bauer, Waldemar

    2014-09-01

    Space debris is a growing problem. Models show that the Kessler syndrome, the exponential growth of debris due to collisions, has become unavoidable unless an active debris removal program is initiated. The debris population in LEO with inclination between 60° and 95° is considered as the most critical zone. In order to stabilize the debris population in orbit, especially in LEO, 5 to 10 objects will need to be removed every year. The unique circumstances of such a mission could require that several objects are removed with a single launch. This will require a mission to rendezvous with a multitude of objects orbiting on different altitudes, inclinations and planes. Removal models have assumed that the top priority targets will be removed first. However this will lead to a suboptimal mission design and increase the ΔV-budget. Since there is a multitude of targets to choose from, the targets can be selected for an optimal mission design. In order to select a group of targets for a removal mission the orbital parameters and political constraints should also be taken into account. Within this paper a number of the target selection criteria are presented. The possible mission targets and their order of retrieval is dependent on the mission architecture. A comparison between several global mission architectures is given. Under consideration are 3 global missions of which a number of parameters are varied. The first mission launches multiple separate deorbit kits. The second launches a mother craft with deorbit kits. The third launches an orbital tug which pulls the debris in a lower orbit, after which a deorbit kit performs the final deorbit burn. A RoM mass and cost comparison is presented. The research described in this paper has been conducted as part of an active debris removal study by the Advanced Study Group (ASG). The ASG is an interdisciplinary student group working at the DLR, analyzing existing technologies and developing new ideas into preliminary concepts.

  17. Target-selective protein S-nitrosylation by sequence motif recognition.

    PubMed

    Jia, Jie; Arif, Abul; Terenzi, Fulvia; Willard, Belinda; Plow, Edward F; Hazen, Stanley L; Fox, Paul L

    2014-10-23

    S-nitrosylation is a ubiquitous protein modification emerging as a principal mechanism of nitric oxide (NO)-mediated signal transduction and cell function. S-nitrosylases can use NO synthase (NOS)-derived NO to modify selected cysteines in target proteins. Despite proteomic identification of over a thousand S-nitrosylated proteins, few S-nitrosylases have been identified. Moreover, mechanisms underlying site-selective S-nitrosylation and the potential role of specific sequence motifs remain largely unknown. Here, we describe a stimulus-inducible, heterotrimeric S-nitrosylase complex consisting of inducible NOS (iNOS), S100A8, and S100A9. S100A9 exhibits transnitrosylase activity, shuttling NO from iNOS to the target protein, whereas S100A8 and S100A9 coordinately direct site selection. A family of proteins S-nitrosylated by iNOS-S100A8/A9 were revealed by proteomic analysis. A conserved I/L-X-C-X2-D/E motif was necessary and sufficient for iNOS-S100A8/A9-mediated S-nitrosylation. These results reveal an elusive parallel between protein S-nitrosylation and phosphorylation, namely, stimulus-dependent posttranslational modification of selected targets by primary sequence motif recognition. PMID:25417112

  18. BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

    PubMed Central

    Setty, Subba Rao Gangi; Tenza, Danile; Truschel, Steven T.; Chou, Evelyn; Sviderskaya, Elena V.; Theos, Alexander C.; Lamoreux, M. Lynn; Di Pietro, Santiago M.; Starcevic, Marta; Bennett, Dorothy C.; Dell'Angelica, Esteban C.; Raposo, Graa

    2007-01-01

    Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS. PMID:17182842

  19. Using Multiplexed Assays of Oncogenic Drivers in Lung Cancers to Select Targeted Drugs

    PubMed Central

    Kris, Mark G.; Johnson, Bruce E.; Berry, Lynne D.; Kwiatkowski, David J.; Iafrate, A. John; Wistuba, Ignacio I.; Varella-Garcia, Marileila; Franklin, Wilbur A.; Aronson, Samuel L.; Su, Pei-Fang; Shyr, Yu; Camidge, D. Ross; Sequist, Lecia V.; Glisson, Bonnie S.; Khuri, Fadlo R.; Garon, Edward B.; Pao, William; Rudin, Charles; Schiller, Joan; Haura, Eric B.; Socinski, Mark; Shirai, Keisuke; Chen, Heidi; Giaccone, Giuseppe; Ladanyi, Marc; Kugler, Kelly; Minna, John D.; Bunn, Paul A.

    2014-01-01

    IMPORTANCE Targeting oncogenic drivers (genomic alterations critical to cancer development and maintenance) has transformed the care of patients with lung adenocarcinomas. The Lung Cancer Mutation Consortium was formed to perform multiplexed assays testing adenocarcinomas of the lung for drivers in 10 genes to enable clinicians to select targeted treatments and enroll patients into clinical trials. OBJECTIVES To determine the frequency of oncogenic drivers in patients with lung adenocarcinomas and to use the data to select treatments targeting the identified driver(s) and measure survival. DESIGN, SETTING, AND PARTICIPANTS From 2009 through 2012, 14 sites in the United States enrolled patients with metastatic lung adenocarcinomas and a performance status of 0 through 2 and tested their tumors for 10 drivers. Information was collected on patients, therapies, and survival. INTERVENTIONS Tumors were tested for 10 oncogenic drivers, and results were used to select matched targeted therapies. MAIN OUTCOMES AND MEASURES Determination of the frequency of oncogenic drivers, the proportion of patients treated with genotype-directed therapy, and survival. RESULTS From 2009 through 2012, tumors from 1007 patients were tested for at least 1 gene and 733 for 10 genes (patients with full genotyping). An oncogenic driver was found in 466 of 733 patients (64%). Among these 733 tumors, 182 tumors (25%) had the KRAS driver; sensitizing EGFR, 122 (17%); ALK rearrangements, 57 (8%); other EGFR, 29 (4%); 2 or more genes, 24 (3%); ERBB2 (formerly HER2), 19 (3%); BRAF, 16 (2%); PIK3CA, 6 (<1%); MET amplification, 5 (<1%); NRAS, 5 (<1%); MEK1, 1 (<1%); AKT1, 0. Results were used to select a targeted therapy or trial in 275 of 1007 patients (28%). The median survival was 3.5 years (interquartile range [IQR], 1.96-7.70) for the 260 patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years (IQR, 0.88-6.20) for the 318 patients with any oncogenic driver(s) who did not receive genotype-directed therapy (propensity scoreadjusted hazard ratio, 0.69 [95% CI, 0.53-0.9], P = .006). CONCLUSIONS AND RELEVANCE Actionable drivers were detected in 64% of lung adenocarcinomas. Multiplexed testing aided physicians in selecting therapies. Although individuals with drivers receiving a matched targeted agent lived longer, randomized trials are required to determine if targeting therapy based on oncogenic drivers improves survival. PMID:24846037

  20. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells

    PubMed Central

    He, Huan; Li, Dong-Wei; Yang, Li-Yun; Fu, Li; Zhu, Xun-Jin; Wong, Wai-Kwok; Jiang, Feng-Lei; Liu, Yi

    2015-01-01

    Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. PMID:26337336

  1. Gene expression levels are a target of recent natural selection in the human genome.

    PubMed

    Kudaravalli, Sridhar; Veyrieras, Jean-Baptiste; Stranger, Barbara E; Dermitzakis, Emmanouil T; Pritchard, Jonathan K

    2009-03-01

    Changes in gene expression may represent an important mode of human adaptation. However, to date, there are relatively few known examples in which selection has been shown to act directly on levels or patterns of gene expression. In order to test whether single nucleotide polymorphisms (SNPs) that affect gene expression in cis are frequently targets of positive natural selection in humans, we analyzed genome-wide SNP and expression data from cell lines associated with the International HapMap Project. Using a haplotype-based test for selection that was designed to detect incomplete selective sweeps, we found that SNPs showing signals of selection are more likely than random SNPs to be associated with gene expression levels in cis. This signal is significant in the Yoruba (which is the population that shows the strongest signals of selection overall) and shows a trend in the same direction in the other HapMap populations. Our results argue that selection on gene expression levels is an important type of human adaptation. Finally, our work provides an analytical framework for tackling a more general problem that will become increasingly important: namely, testing whether selection signals overlap significantly with SNPs that are associated with phenotypes of interest. PMID:19091723

  2. Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

    PubMed Central

    Vannucci, Luca; Falvo, Elisabetta; Fornara, Manuela; Di Micco, Patrizio; Benada, Oldrich; Krizan, Jiri; Svoboda, Jan; Hulikova-Capkova, Katarina; Morea, Veronica; Boffi, Alberto; Ceci, Pierpaolo

    2012-01-01

    Background Nanoparticle-based systems are promising for the development of imaging and therapeutic agents. The main advantage of nanoparticles over traditional systems lies in the possibility of loading multiple functionalities onto a single molecule, which are useful for therapeutic and/or diagnostic purposes. These functionalities include targeting moieties which are able to recognize receptors overexpressed by specific cells and tissues. However, targeted delivery of nanoparticles requires an accurate system design. We present here a rationally designed, genetically engineered, and chemically modified protein-based nanoplatform for cell/tissue-specific targeting. Methods Our nanoparticle constructs were based on the heavy chain of the human protein ferritin (HFt), a highly symmetrical assembly of 24 subunits enclosing a hollow cavity. HFt-based nanoparticles were produced using both genetic engineering and chemical functionalization methods to impart several functionalities, ie, the α-melanocyte-stimulating hormone peptide as a melanoma-targeting moiety, stabilizing and HFt-masking polyethylene glycol molecules, rhodamine fluorophores, and magnetic resonance imaging agents. The constructs produced were extensively characterized by a number of physicochemical techniques, and assayed for selective melanoma-targeting in vitro and in vivo. Results Our HFt-based nanoparticle constructs functionalized with the α-melanocyte-stimulating hormone peptide moiety and polyethylene glycol molecules were specifically taken up by melanoma cells but not by other cancer cell types in vitro. Moreover, experiments in melanoma-bearing mice indicate that these constructs have an excellent tumor-targeting profile and a long circulation time in vivo. Conclusion By masking human HFt with polyethylene glycol and targeting it with an α-melanocyte-stimulating hormone peptide, we developed an HFt-based melanoma-targeting nanoplatform for application in melanoma diagnosis and treatment. These results could be of general interest, because the same strategy can be exploited to develop ad hoc nanoplatforms for specific delivery towards any cell/tissue type for which a suitable targeting moiety is available. PMID:22619508

  3. Increased lysosomal biogenesis in activated microglia and exacerbated neuronal damage after traumatic brain injury in progranulin-deficient mice.

    PubMed

    Tanaka, Y; Matsuwaki, T; Yamanouchi, K; Nishihara, M

    2013-10-10

    Progranulin (PGRN) is known to play a role in the pathogenesis of neurodegenerative diseases. Recently, it has been demonstrated that patients with the homozygous mutation in the GRN gene present with neuronal ceroid lipofuscinosis, and there is growing evidence that PGRN is related to lysosomal function. In the present study, we investigated the possible role of PGRN in the lysosomes of activated microglia in the cerebral cortex after traumatic brain injury (TBI). We showed that the mouse GRN gene has two possible coordinated lysosomal expression and regulation (CLEAR) sequences that bind to transcription factor EB (TFEB), a master regulator of lysosomal genes. PGRN was colocalized with Lamp1, a lysosomal marker, and Lamp1-positive areas in GRN-deficient (KO) mice were significantly expanded compared with wild-type (WT) mice after TBI. Expression of all the lysosome-related genes examined in KO mice was significantly higher than that in WT mice. The number of activated microglia with TFEB localized to the nucleus was also significantly increased in KO as compared with WT mice. Since the TFEB translocation is regulated by the mammalian target of rapamycin complex 1 (mTORC1) activity in the lysosome, we compared ribosomal S6 kinase 1 (S6K1) phosphorylation that reflects mTORC1 activity. S6K1 phosphorylation in KO mice was significantly lower than that in WT mice. In addition, the number of nissl-positive and fluoro-jade B-positive cells around the injury was significantly decreased and increased, respectively, in KO as compared with WT mice. These results suggest that PGRN localized in the lysosome is involved in the activation of mTORC1, and its deficiency leads to increased TFEB nuclear translocation with a resultant increase in lysosomal biogenesis in activated microglia and exacerbated neuronal damage in the cerebral cortex after TBI. PMID:23830905

  4. Lysosomal pH Plays a Key Role in Regulation of mTOR Activity in Osteoclasts.

    PubMed

    Hu, Yingwei; Carraro-Lacroix, Luciene R; Wang, Andrew; Owen, Celeste; Bajenova, Elena; Corey, Paul N; Brumell, John H; Voronov, Irina

    2016-02-01

    Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth. It has been shown to play an important role in osteoclast differentiation, particularly at the earlier stages of osteoclastogenesis. mTOR activation and function, as part of mTORC1 complex, is dependent on lysosomal localization and the vacuolar H(+) -ATPase (V-ATPase) activity; however, the precise mechanism is still not well understood. Using primary mouse osteoclasts that are known to have higher lysosomal pH due to R740S mutation in the V-ATPase a3 subunit, we investigated the role of lysosomal pH in mTORC1 signaling. Our results demonstrated that +/R740S cells had increased basal mTOR protein levels and mTORC1 activity compared to +/+ osteoclasts, while mTOR gene expression was decreased. Treatment with lysosomal inhibitors chloroquine and ammonium chloride, compounds known to raise lysosomal pH, significantly increased mTOR protein levels in +/+ cells, confirming the importance of lysosomal pH in mTOR signaling. These results also suggested that mTOR could be degraded in the lysosome. To test this hypothesis, we cultured osteoclasts with chloroquine or proteasomal inhibitor MG132. Both chloroquine and MG132 increased mTOR and p-mTOR protein levels in +/+ osteoclasts, suggesting that mTOR undergoes both lysosomal and proteasomal degradation. Treatment with cycloheximide, an inhibitor of new protein synthesis, confirmed that mTOR is constitutively expressed and degraded. These results show that, in osteoclasts, the lysosome plays a key role not only in mTOR activation but also in its deactivation through protein degradation, representing a novel molecular mechanism of mTOR regulation. J. Cell. Biochem. 117: 413-425, 2016. 2015 Wiley Periodicals, Inc. PMID:26212375

  5. A new method to select aimpoint for airplane target at end term

    NASA Astrophysics Data System (ADS)

    Li, Lijuan

    2013-10-01

    Selecting key point in airplane target as tracking aimpoint at end term is important for IR imaging missiles to improve guidance accuracy. A new aimpoint selection method proper for engineering application is proposed in this article. Other than tracking the center of plume which is the most marked property of airplanes, some point near engine is selected as aimpoint. Firstly plume and skin are extracted by using different thresholds according to their gray scale statistics and features like circularity, distance ratio and central axis are obtained to classify the image types. Then referring to these image types, the centroid of the segmented sector or a point on the line of central axis of plume sector are selected as aimpoint respectively. The algorithm has the advantage of more efficiency in both space and time consuming. Tests have shown the validity of the algorithm.

  6. Criteria for dendritic cell receptor selection for efficient antibody-targeted vaccination.

    PubMed

    Reuter, Anika; Panozza, Scott E; Macri, Christophe; Dumont, Claire; Li, Jessica; Liu, Haiyin; Segura, Elodie; Vega-Ramos, Javier; Gupta, Nishma; Caminschi, Irina; Villadangos, Jose A; Johnston, Angus P R; Mintern, Justine D

    2015-03-15

    Ab-targeted vaccination involves targeting a receptor of choice expressed by dendritic cells (DCs) with Ag-coupled Abs. Currently, there is little consensus as to which criteria determine receptor selection to ensure superior Ag presentation and immunity. In this study, we investigated parameters of DC receptor internalization and determined how they impact Ag presentation outcomes. First, using mixed bone marrow chimeras, we established that Ag-targeted, but not nontargeted, DCs are responsible for Ag presentation in settings of Ab-targeted vaccination in vivo. Next, we analyzed parameters of DEC205 (CD205), Clec9A, CD11c, CD11b, and CD40 endocytosis and obtained quantitative measurements of internalization speed, surface turnover, and delivered Ag load. Exploiting these parameters in MHC class I (MHC I) and MHC class II (MHC II) Ag presentation assays, we showed that receptor expression level, proportion of surface turnover, or speed of receptor internalization did not impact MHC I or MHC II Ag presentation efficiency. Furthermore, the Ag load delivered to DCs did not correlate with the efficiency of MHC I or MHC II Ag presentation. In contrast, targeting Ag to CD8(+) or CD8(-) DCs enhanced MHC I or MHC II Ag presentation, respectively. Therefore, receptor expression levels, speed of internalization, and/or the amount of Ag delivered can be excluded as major determinants that dictate Ag presentation efficiency in setting of Ab-targeted vaccination. PMID:25653426

  7. Masking and triggered unmasking of targeting ligands on liposomal chemotherapy selectively suppress tumor growth in vivo.

    PubMed

    Bandekar, Amey; Zhu, Charles; Gomez, Ana; Menzenski, Monica Zofia; Sempkowski, Michelle; Sofou, Stavroula

    2013-01-01

    We investigated the feasibility and efficacy of a drug delivery strategy to vascularized cancer that combines targeting selectivity with high uptake by targeted cells and high bioexposure of cells to delivered chemotherapeutics. Targeted lipid vesicles composed of pH responsive membranes were designed to reversibly form phase-separated lipid domains, which are utilized to tune the vesicle's apparent functionality and permeability. During circulation, vesicles mask functional ligands and stably retain their contents. Upon extravasation in the tumor interstitium, ligand-labeled lipids become unmasked and segregated within lipid domains triggering targeting to cancer cells followed by internalization. In the acidic endosome, vesicles burst release the encapsulated therapeutics through leaky boundaries around the phase-separated lipid domains. The pH tunable vesicles contain doxorubicin and are labeled with an anti-HER2 peptide. In vitro, anti-HER2 pH tunable vesicles release doxorubicin in a pH dependent manner, and exhibit 233% increase in binding to HER2-overexpressing BT474 breast cancer cells with lowering pH from 7.4 to 6.5 followed by significant (50%) internalization. In subcutaneous BT474 xenografts in nude mice, targeted pH tunable vesicles decrease tumor volumes by 159% relative to nontargeted vesicles, and they also exhibit better tumor control by 11% relative to targeted vesicles without an unmasking property. These results suggest the potential of pH tunable vesicles to ultimately control tumor growth at relatively lower administered doses. PMID:23134440

  8. Application of support vector machine-based ranking strategies to search for target-selective compounds.

    PubMed

    Wassermann, Anne Mai; Geppert, Hanna; Bajorath, Jürgen

    2011-01-01

    Support vector machine (SVM)-based selectivity searching has recently been introduced to identify compounds in virtual screening libraries that are not only active for a target protein, but also selective for this target over a closely related member of the same protein family. In simulated virtual screening calculations, SVM-based strategies termed preference ranking and one-versus-all ranking were successfully applied to rank a database and enrich high-ranking positions with selective compounds while removing nonselective molecules from high ranks. In contrast to the original SVM approach developed for binary classification, these strategies enable learning from more than two classes, considering that distinguishing between selective, promiscuously active, and inactive compounds gives rise to a three-class prediction problem. In this chapter, we describe the extension of the one-versus-all strategy to four training classes. Furthermore, we present an adaptation of the preference ranking strategy that leads to higher recall of selective compounds than previously investigated approaches and is applicable in situations where the removal of nonselective compounds from high-ranking positions is not required. PMID:20838983

  9. Close encounters of the lysosome/peroxisome kind

    PubMed Central

    Jin, Yui; Strunk, Bethany S.; Weisman, Lois S.

    2015-01-01

    Lysosomes provide a major source for cellular cholesterol; however, most of this cholesterol is trafficked to the plasma membrane via unknown mechanisms. In this issue of Cell, Chu et al. identify an unexpected role for peroxisomes in the transport of cholesterol from the lysosome to the plasma membrane via a lysosome-peroxisome membrane contact site. PMID:25860602

  10. Lorcaserin and pimavanserin: emerging selectivity of serotonin receptor subtypetargeted drugs

    PubMed Central

    Meltzer, Herbert Y.; Roth, Bryan L.

    2013-01-01

    Serotonin (5-hydroxytryptamine, or 5-HT) receptors mediate a plethora of physiological phenomena in the brain and the periphery. Additionally, serotonergic dysfunction has been implicated in nearly every neuropsychiatric disorder. The effects of serotonin are mediated by fourteen GPCRs. Both the therapeutic actions and side effects of commonly prescribed drugs are frequently due to nonspecific actions on various 5-HT receptor subtypes. For more than 20 years, the search for clinically efficacious drugs that selectively target 5-HT receptor subtypes has been only occasionally successful. This review provides an overview of 5-HT receptor pharmacology and discusses two recent 5-HT receptor subtypeselective drugs, lorcaserin and pimavanserin, which target the 5HT2C and 5HT2A receptors and provide new treatments for obesity and Parkinsons disease psychosis, respectively. PMID:24292660

  11. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors.

    PubMed

    Ojo, Kayode K; Larson, Eric T; Keyloun, Katelyn R; Castaneda, Lisa J; Derocher, Amy E; Inampudi, Krishna K; Kim, Jessica E; Arakaki, Tracy L; Murphy, Ryan C; Zhang, Li; Napuli, Alberto J; Maly, Dustin J; Verlinde, Christophe L M J; Buckner, Frederick S; Parsons, Marilyn; Hol, Wim G J; Merritt, Ethan A; Van Voorhis, Wesley C

    2010-05-01

    New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable. PMID:20436472

  12. Impact of high-risk conjunctions on Active Debris Removal target selection

    NASA Astrophysics Data System (ADS)

    Lidtke, Aleksander A.; Lewis, Hugh G.; Armellin, Roberto

    2015-10-01

    Space debris simulations show that if current space launches continue unchanged, spacecraft operations might become difficult in the congested space environment. It has been suggested that Active Debris Removal (ADR) might be necessary in order to prevent such a situation. Selection of objects to be targeted by ADR is considered important because removal of non-relevant objects will unnecessarily increase the cost of ADR. One of the factors to be used in this ADR target selection is the collision probability accumulated by every object. This paper shows the impact of high-probability conjunctions on the collision probability accumulated by individual objects as well as the probability of any collision occurring in orbit. Such conjunctions cannot be predicted far in advance and, consequently, not all the objects that will be involved in such dangerous conjunctions can be removed through ADR. Therefore, a debris remediation method that would address such events at short notice, and thus help prevent likely collisions, is suggested.

  13. Designing the nanobiointerface of fluorescent nanodiamonds: highly selective targeting of glioma cancer cells

    NASA Astrophysics Data System (ADS)

    Slegerova, Jitka; Hajek, Miroslav; Rehor, Ivan; Sedlak, Frantisek; Stursa, Jan; Hruby, Martin; Cigler, Petr

    2014-12-01

    Core-shell nanoparticles based on fluorescent nanodiamonds coated with a biocompatible N-(2-hydroxypropyl)methacrylamide copolymer shell were developed for background-free near-infrared imaging of cancer cells. The particles showed excellent colloidal stability in buffers and culture media. After conjugation with a cyclic RGD peptide they selectively targeted integrin ?v?3 receptors on glioblastoma cells with high internalization efficacy.Core-shell nanoparticles based on fluorescent nanodiamonds coated with a biocompatible N-(2-hydroxypropyl)methacrylamide copolymer shell were developed for background-free near-infrared imaging of cancer cells. The particles showed excellent colloidal stability in buffers and culture media. After conjugation with a cyclic RGD peptide they selectively targeted integrin ?v?3 receptors on glioblastoma cells with high internalization efficacy. Electronic supplementary information (ESI) available: Materials and methods, colloidal stability studies and cell viability studies. See DOI: 10.1039/c4nr02776k

  14. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

    PubMed Central

    Corrotte, M.; Castro-Gomes, T.; Koushik, A.B.; Andrews, N.W.

    2016-01-01

    Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as ChediakHigashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and NiemannPick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane. PMID:25665445

  15. A RANKL–PKCβ–TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts

    PubMed Central

    Ferron, Mathieu; Settembre, Carmine; Shimazu, Junko; Lacombe, Julie; Kato, Shigeaki; Rawlings, David J.; Ballabio, Andrea; Karsenty, Gerard

    2013-01-01

    Bone resorption by osteoclasts requires a large number of lysosomes that release proteases in the resorption lacuna. Whether lysosomal biogenesis is a consequence of the action of transcriptional regulators of osteoclast differentiation or is under the control of a different and specific transcriptional pathway remains unknown. We show here, through cell-based assays and cell-specific gene deletion experiments in mice, that the osteoclast differentiation factor RANKL promotes lysosomal biogenesis once osteoclasts are differentiated through the selective activation of TFEB, a member of the MITF/TFE family of transcription factors. This occurs following PKCβ phosphorylation of TFEB on three serine residues located in its last 15 amino acids. This post-translational modification stabilizes and increases the activity of this transcription factor. Supporting these biochemical observations, mice lacking in osteoclasts—either TFEB or PKCβ—show decreased lysosomal gene expression and increased bone mass. Altogether, these results uncover a RANKL-dependent signaling pathway taking place in differentiated osteoclasts and culminating in the activation of TFEB to enhance lysosomal biogenesis—a necessary step for proper bone resorption. PMID:23599343

  16. Microfluidics for Drug Discovery and Development: From Target Selection to Product Lifecycle Management

    PubMed Central

    Kang, Lifeng; Chung, Bong Geun; Langer, Robert; Khademhosseini, Ali

    2009-01-01

    Microfluidic technologies’ ability to miniaturize assays and increase experimental throughput have generated significant interest in the drug discovery and development domain. These characteristics make microfluidic systems a potentially valuable tool for many drug discovery and development applications. Here, we review the recent advances of microfluidic devices for drug discovery and development and highlight their applications in different stages of the process, including target selection, lead identification, preclinical tests, clinical trials, chemical synthesis, formulations studies, and product management. PMID:18190858

  17. Spectral selective radio frequency emissions from laser induced breakdown of target materials

    SciTech Connect

    Vinoth Kumar, L.; Manikanta, E.; Leela, Ch.; Prem Kiran, P.

    2014-08-11

    The radio frequency emissions scanned over broad spectral range (30?MHz1?GHz) from single shot nanosecond (7?ns) and picosecond (30 ps) laser induced breakdown (LIB) of different target materials (atmospheric air, aluminum, and copper) are presented. The dominant emissions from ns-LIB, compared to those from the ps-LIB, indicate the presence and importance of atomic and molecular clusters in the plasma. The dynamics of laser pulse-matter interaction and the properties of the target materials were found to play an important role in determining the plasma parameters which subsequently determine the emissions. Thus, with a particular laser and target material, the emissions were observed to be spectral selective. The radiation detection capability was observed to be relatively higher, when the polarization of the input laser and the antenna is same.

  18. Mitochondrial permeability transition pore as a selective target for anti-cancer therapy

    PubMed Central

    Suh, Dong H.; Kim, Mi-Kyung; Kim, Hee S.; Chung, Hyun H.; Song, Yong S.

    2012-01-01

    Mitochondrial outer membrane permeabilization (MOMP) is the ultimate step in dozens of lethal apoptotic signal transduction pathways which converge on mitochondria. One of the representative systems proposed to be responsible for the MOMP is the mitochondrial permeability transition pore (MPTP). Although the molecular composition of the MPTP is not clearly understood, the MPTP attracts much interest as a promising target for resolving two conundrums regarding cancer treatment: tumor selectivity and resistance to treatment. The regulation of the MPTP is closely related to metabolic reprogramming in cancer cells including mitochondrial alterations. Restoration of deregulated apoptotic machinery in cancer cells by tumor-specific modulation of the MPTP could therefore be a promising anti-cancer strategy. Currently, a number of MPTP-targeting agents are under pre-clinical and clinical studies. Here, we reviewed the structure and regulation of the MPTP as well as the current status of the development of promising MPTP-targeting drugs. PMID:23483560

  19. Functionalized paper SERS (P-SERS) substrates for selective targeting of analytes in complex samples

    NASA Astrophysics Data System (ADS)

    Yu, Wei W.; Hoppmann, Eric P.

    2015-05-01

    Surface enhanced Raman spectroscopy (SERS) requires the analyte molecule to be close to the plasmonic surface in order to generate SERS enhancement. This limitation restricts the practical application of SERS to molecules that possess functional groups that interact strongly with gold or silver surfaces. Moreover, the identification of target analytes in a complex sample matrix is made even more difficult when interferents compete with the target for binding to the plasmonic surface, resulting in overlapping spectral signatures. In this work, we report a strategy to functionalize inkjet printed P-SERS substrates by strategically placing supramolecular structures (such as nucleic acid aptamers) onto the gold nanoparticles. This promotes the selective interaction of target molecules with the plasmonic surface, leading to improved sensor performance.

  20. Photostick: a method for selective isolation of target cells from culture

    PubMed Central

    Chien, Miao-Ping; Werley, Christopher A.; Farhi, Samouil L.

    2015-01-01

    Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The photostick technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns. PMID:25705368

  1. Lysosomal Integral Membrane Protein-2: A New Player in Lysosome-Related Pathology

    PubMed Central

    Gonzalez, Ashley; Valeiras, Mark; Sidransky, Ellen; Tayebi, Nahid

    2014-01-01

    Lysosomes require the presence of many specialized proteins to facilitate their roles in cellular maintenance. One such protein that has proven to be an important player in the lysosomal field is lysosomal integral membrane protein-2 (LIMP-2), encoded by the gene SCARB2. LIMP-2 is required for the normal biogenesis and maintenance of lysosomes and endosomes and has been identified as the specific receptor for glucocerebrosidase, the enzyme deficient in Gaucher disease. Research into LIMP-2 and the SCARB2 gene indicate that it may be a factor contributing to the clinical heterogeneity seen among patients with Gaucher disease. Mutations in SCARB2 have also been identified as the cause of action myoclonus renal failure (AMRF), and in some cases progressive myoclonic epilepsy. A total of 14 disease-causing SCARB2 mutations have been identified to date. The role of LIMP-2 in human pathology has expanded with its identification as a component of the intercalated disc in cardiac muscle and as a receptor for specific enteroviruses, two unanticipated findings that reaffirm the myriad roles of lysosomal proteins. Studies into the full impact of LIMP-2 deficiency and the LIMP2/glucocerebrosidase molecular pathway will lead to a better understanding of disease pathogenesis in Gaucher disease and AMRF, and to new insights into lysosomal processing, trafficking and function. PMID:24389070

  2. Targeting hunter distribution based on host resource selection and kill sites to manage disease risk.

    PubMed

    Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K

    2013-10-01

    Endemic and emerging diseases are rarely uniform in their spatial distribution or prevalence among cohorts of wildlife. Spatial models that quantify risk-driven differences in resource selection and hunter mortality of animals at fine spatial scales can assist disease management by identifying high-risk areas and individuals. We used resource selection functions (RSFs) and selection ratios (SRs) to quantify sex- and age-specific resource selection patterns of collared (n = 67) and hunter-killed (n = 796) nonmigratory elk (Cervus canadensis manitobensis) during the hunting season between 2002 and 2012, in southwestern Manitoba, Canada. Distance to protected area was the most important covariate influencing resource selection and hunter-kill sites of elk (AICw = 1.00). Collared adult males (which are most likely to be infected with bovine tuberculosis (Mycobacterium bovis) and chronic wasting disease) rarely selected for sites outside of parks during the hunting season in contrast to adult females and juvenile males. The RSFs showed selection by adult females and juvenile males to be negatively associated with landscape-level forest cover, high road density, and water cover, whereas hunter-kill sites of these cohorts were positively associated with landscape-level forest cover and increasing distance to streams and negatively associated with high road density. Local-level forest was positively associated with collared animal locations and hunter-kill sites; however, selection was stronger for collared juvenile males and hunter-killed adult females. In instances where disease infects a metapopulation and eradication is infeasible, a principle goal of management is to limit the spread of disease among infected animals. We map high-risk areas that are regularly used by potentially infectious hosts but currently underrepresented in the distribution of kill sites. We present a novel application of widely available data to target hunter distribution based on host resource selection and kill sites as a promising tool for applying selective hunting to the management of transmissible diseases in a game species. PMID:24324876

  3. Targeting hunter distribution based on host resource selection and kill sites to manage disease risk

    PubMed Central

    Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K

    2013-01-01

    Endemic and emerging diseases are rarely uniform in their spatial distribution or prevalence among cohorts of wildlife. Spatial models that quantify risk-driven differences in resource selection and hunter mortality of animals at fine spatial scales can assist disease management by identifying high-risk areas and individuals. We used resource selection functions (RSFs) and selection ratios (SRs) to quantify sex- and age-specific resource selection patterns of collared (n = 67) and hunter-killed (n = 796) nonmigratory elk (Cervus canadensis manitobensis) during the hunting season between 2002 and 2012, in southwestern Manitoba, Canada. Distance to protected area was the most important covariate influencing resource selection and hunter-kill sites of elk (AICw = 1.00). Collared adult males (which are most likely to be infected with bovine tuberculosis (Mycobacterium bovis) and chronic wasting disease) rarely selected for sites outside of parks during the hunting season in contrast to adult females and juvenile males. The RSFs showed selection by adult females and juvenile males to be negatively associated with landscape-level forest cover, high road density, and water cover, whereas hunter-kill sites of these cohorts were positively associated with landscape-level forest cover and increasing distance to streams and negatively associated with high road density. Local-level forest was positively associated with collared animal locations and hunter-kill sites; however, selection was stronger for collared juvenile males and hunter-killed adult females. In instances where disease infects a metapopulation and eradication is infeasible, a principle goal of management is to limit the spread of disease among infected animals. We map high-risk areas that are regularly used by potentially infectious hosts but currently underrepresented in the distribution of kill sites. We present a novel application of widely available data to target hunter distribution based on host resource selection and kill sites as a promising tool for applying selective hunting to the management of transmissible diseases in a game species. PMID:24324876

  4. Synthetic, Non-saccharide, Glycosaminoglycan Mimetics Selectively Target Colon Cancer Stem Cells

    PubMed Central

    2015-01-01

    Selective targeting of cancer stem-like cells (CSCs) is a paradigm-shifting approach. We hypothesized that CSCs can be targeted by interfering with functions of sulfated glycosaminoglycans, which play key roles in cancer cell growth, invasion and metastasis. We developed a tandem, dual screen strategy involving (1) assessing inhibition of monolayer versus spheroid growth and (2) assessing inhibition of primary versus secondary spheroid growth to identify G2.2, a unique sulfated nonsaccharide GAG mimetic (NSGM) from a focused library of 53 molecules, as a selective inhibitor of colon CSCs. The NSGM down-regulated several CSC markers through regulation of gene transcription, while closely related, inactive NSGMs G1.4 and G4.1 demonstrated no such changes. G2.2s effects on CSCs were mediated, in part, through induction of apoptosis and inhibition of self-renewal factors. Overall, this work presents the proof-of-principle that CSCs can be selectively targeted through novel NSGMs, which are likely to advance fundamental understanding on CSCs while also aiding development of novel therapeutic agents. PMID:24968014

  5. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    PubMed

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. PMID:26627837

  6. THE SDSS-III BARYON OSCILLATION SPECTROSCOPIC SURVEY: QUASAR TARGET SELECTION FOR DATA RELEASE NINE

    SciTech Connect

    Ross, Nicholas P.; Kirkpatrick, Jessica A.; Carithers, William C.; Ho, Shirley; Myers, Adam D.; Sheldon, Erin S.; Yeche, Christophe; Aubourg, Eric; Strauss, Michael A.; Lee, Khee-Gan; Bovy, Jo; Blanton, Michael R.; Hogg, David W.; Richards, Gordon T.; Brandt, W. N.; Croft, Rupert A. C.; Da Silva, Robert; Dawson, Kyle; Eisenstein, Daniel J.; Hennawi, Joseph F.; and others

    2012-03-01

    The SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS), a five-year spectroscopic survey of 10,000 deg{sup 2}, achieved first light in late 2009. One of the key goals of BOSS is to measure the signature of baryon acoustic oscillations (BAOs) in the distribution of Ly{alpha} absorption from the spectra of a sample of {approx}150,000 z > 2.2 quasars. Along with measuring the angular diameter distance at z Almost-Equal-To 2.5, BOSS will provide the first direct measurement of the expansion rate of the universe at z > 2. One of the biggest challenges in achieving this goal is an efficient target selection algorithm for quasars in the redshift range 2.2 < z < 3.5, where their colors tend to overlap those of the far more numerous stars. During the first year of the BOSS survey, quasar target selection (QTS) methods were developed and tested to meet the requirement of delivering at least 15 quasars deg{sup -2} in this redshift range, with a goal of 20 out of 40 targets deg{sup -2} allocated to the quasar survey. To achieve these surface densities, the magnitude limit of the quasar targets was set at g {<=} 22.0 or r {<=} 21.85. While detection of the BAO signature in the distribution of Ly{alpha} absorption in quasar spectra does not require a uniform target selection algorithm, many other astrophysical studies do. We have therefore defined a uniformly selected subsample of 20 targets deg{sup -2}, for which the selection efficiency is just over 50% ({approx}10 z > 2.20 quasars deg{sup -2}). This 'CORE' subsample will be fixed for Years Two through Five of the survey. For the remaining 20 targets deg{sup -2}, we will continue to develop improved selection techniques, including the use of additional data sets beyond the Sloan Digital Sky Survey (SDSS) imaging data. In this paper, we describe the evolution and implementation of the BOSS QTS algorithms during the first two years of BOSS operations (through 2011 July), in support of the science investigations based on these data, and we analyze the spectra obtained during the first year. During this year, 11,263 new z > 2.20 quasars were spectroscopically confirmed by BOSS, roughly double the number of previously known quasars with z > 2.20. Our current algorithms select an average of 15 z > 2.20 quasars deg{sup -2} from 40 targets deg{sup -2} using single-epoch SDSS imaging. Multi-epoch optical data and data at other wavelengths can further improve the efficiency and completeness of BOSS QTS.

  7. Target Selection and Deselection at the Berkeley StructuralGenomics Center

    SciTech Connect

    Chandonia, John-Marc; Kim, Sung-Hou; Brenner, Steven E.

    2005-03-22

    At the Berkeley Structural Genomics Center (BSGC), our goalis to obtain a near-complete structural complement of proteins in theminimal organisms Mycoplasma genitalium and M. pneumoniae, two closelyrelated pathogens. Current targets for structure determination have beenselected in six major stages, starting with those predicted to be mosttractable to high throughput study and likely to yield new structuralinformation. We report on the process used to select these proteins, aswell as our target deselection procedure. Target deselection reducesexperimental effort by eliminating targets similar to those recentlysolved by the structural biology community or other centers. We measurethe impact of the 69 structures solved at the BSGC as of July 2004 onstructure prediction coverage of the M. pneumoniae and M. genitaliumproteomes. The number of Mycoplasma proteins for which thefold couldfirst be reliably assigned based on structures solved at the BSGC (24 M.pneumoniae and 21 M. genitalium) is approximately 25 percent of the totalresulting from work at all structural genomics centers and the worldwidestructural biology community (94 M. pneumoniae and 86M. genitalium)during the same period. As the number of structures contributed by theBSGC during that period is less than 1 percent of the total worldwideoutput, the benefits of a focused target selection strategy are apparent.If the structures of all current targets were solved, the percentage ofM. pneumoniae proteins for which folds could be reliably assigned wouldincrease from approximately 57 percent (391 of 687) at present to around80 percent (550 of 687), and the percentage of the proteome that could beaccurately modeled would increase from around 37 percent (254 of 687) toabout 64 percent (438 of 687). In M. genitalium, the percentage of theproteome that could be structurally annotated based on structures of ourremaining targets would rise from 72 percent (348 of 486) to around 76percent (371 of 486), with the percentage of accurately modeled proteinswould rise from 50 percent (243 of 486) to 58 percent (283 of 486).Sequences and data on experimental progress on our targets are availablein the public databases Target DB and PEPCdb.

  8. Implications of structural genomics target selection strategies: Pfam5000, whole genome, and random approaches

    SciTech Connect

    Chandonia, John-Marc; Brenner, Steven E.

    2004-07-14

    The structural genomics project is an international effort to determine the three-dimensional shapes of all important biological macromolecules, with a primary focus on proteins. Target proteins should be selected according to a strategy which is medically and biologically relevant, of good value, and tractable. As an option to consider, we present the Pfam5000 strategy, which involves selecting the 5000 most important families from the Pfam database as sources for targets. We compare the Pfam5000 strategy to several other proposed strategies that would require similar numbers of targets. These include including complete solution of several small to moderately sized bacterial proteomes, partial coverage of the human proteome, and random selection of approximately 5000 targets from sequenced genomes. We measure the impact that successful implementation of these strategies would have upon structural interpretation of the proteins in Swiss-Prot, TrEMBL, and 131 complete proteomes (including 10 of eukaryotes) from the Proteome Analysis database at EBI. Solving the structures of proteins from the 5000 largest Pfam families would allow accurate fold assignment for approximately 68 percent of all prokaryotic proteins (covering 59 percent of residues) and 61 percent of eukaryotic proteins (40 percent of residues). More fine-grained coverage which would allow accurate modeling of these proteins would require an order of magnitude more targets. The Pfam5000 strategy may be modified in several ways, for example to focus on larger families, bacterial sequences, or eukaryotic sequences; as long as secondary consideration is given to large families within Pfam, coverage results vary only slightly. In contrast, focusing structural genomics on a single tractable genome would have only a limited impact in structural knowledge of other proteomes: a significant fraction (about 30-40 percent of the proteins, and 40-60 percent of the residues) of each proteome is classified in small families, which may have little overlap with other species of interest. Random selection of targets from one or more genomes is similar to the Pfam5000 strategy in that proteins from larger families are more likely to be chosen, but substantial effort would be spent on small families.

  9. Selective targeting of the retinal pigment epithelium using an acousto-optic laser scanner.

    PubMed

    Alt, Clemens; Framme, Carsten; Schnell, Susanne; Lee, Ho; Brinkmann, Ralf; Lin, Charles P

    2005-01-01

    Selective targeting of the retinal pigment epithelium (RPE) is a new strategy for treating certain retinal disorders while preserving adjacent photoreceptors. The treatment currently relies on a complex laser system to produce the required microsecond pulse structure. In our new approach, we scan the focus of a continuous-wave (cw) laser beam with acousto-optic deflectors to produce microsecond-long exposures at each RPE cell. Experiments were performed in vitro with a bench-top scanner on samples of young bovine RPE and in vivo on Dutch belted rabbits with a slit-lamp adapted scanner. Effective dose 50% (ED50) for RPE damage was determined in vitro by fluorescence cell viability assay and in vivo by fluorescein angiography. Damage to individual RPE cells was achieved with laser power on the order of 100 mW. Using separated scan lines, we demonstrate selectivity in the form of alternating lines of dead and surviving cells that resemble the scan pattern. Selectivity is also shown by the absence of retinal thermal coagulation in vivo. Selective RPE damage is feasible by rapidly scanning a cw laser beam. The scanning device is an attractive alternative to conventional laser coagulation and pulsed laser targeting of the RPE. PMID:16409079

  10. Secondary mAb--vcMMAE conjugates are highly sensitive reporters of antibody internalization via the lysosome pathway.

    PubMed

    Klussman, Kerry; Mixan, Bruce J; Cerveny, Charles G; Meyer, Damon L; Senter, Peter D; Wahl, Alan F

    2004-01-01

    Monoclonal antibodies (mAb) selectively recognizing tumor surface antigens are an important and evolving approach to targeted cancer therapy. One application of therapeutic mAbs is drug targeting via mAb-drug conjugate (ADC) technology. Identification of mAbs capable of internalizing following antigen binding has been accomplished by tracking decline of surface-bound mAb or by internalization of a secondary mAb linked to a toxin. These methods may not be sufficiently sensitive for screening nor wholly predictive of the mAbs' capacity for a specific drug delivery. We have developed a highly selective and sensitive method to detect mAbs for cell internalization and drug delivery. This system uses secondary anti-human or anti-murine mAbs conjugated to the high-potency drug monomethyl auristatin E (MMAE) via a highly stable, enzymatically cleavable linker. Prior studies of this drug linker technology demonstrated internalization of a primary ADC leads to trafficking to lysosomes, drug release by lysosomal cathepsin B, and ensuing cell death. A secondary antibody--drug conjugate (2 degrees ADC) capable of binding primary mAbs bound to the surface of antigen-positive cells has comparable drug delivery capability. The system is sufficiently sensitive to detect internalizing mAbs in nonclonal hybridoma supernatants and is predictive of the activity of subsequently produced primary ADC. Because of their high extracellular stability, the noninternalized 2 degrees ADC are 100--1000-fold less toxic to cells over extended periods of time, permitting an assay in which components can be added without need for separate wash steps. This homogeneous screening system is amenable to medium-throughput screening applications and enables the early identification of mAbs capable of intracellular trafficking for drug delivery and release. PMID:15264863

  11. Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease

    PubMed Central

    Repanas, Kostas; Zingler, Nora; Layer, Liliana E.; Schumann, Gerald G.; Perrakis, Anastassis; Weichenrieder, Oliver

    2007-01-01

    The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for ?28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. Based on the crystal structure, we generated L1-EN mutants to analyze and manipulate DNA target site recognition. Crystal structures and their dynamic and functional analysis show entire loop grafts to be feasible, resulting in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. Structural parameters of the DNA target seem more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons. PMID:17626046

  12. A two-step target binding and selectivity support vector machines approach for virtual screening of dopamine receptor subtype-selective ligands.

    PubMed

    Zhang, Jingxian; Han, Bucong; Wei, Xiaona; Tan, Chunyan; Chen, Yuzong; Jiang, Yuyang

    2012-01-01

    Target selective drugs, such as dopamine receptor (DR) subtype selective ligands, are developed for enhanced therapeutics and reduced side effects. In silico methods have been explored for searching DR selective ligands, but encountered difficulties associated with high subtype similarity and ligand structural diversity. Machine learning methods have shown promising potential in searching target selective compounds. Their target selective capability can be further enhanced. In this work, we introduced a new two-step support vector machines target-binding and selectivity screening method for searching DR subtype-selective ligands, which was tested together with three previously-used machine learning methods for searching D1, D2, D3 and D4 selective ligands. It correctly identified 50.6%-88.0% of the 21-408 subtype selective and 71.7%-81.0% of the 39-147 multi-subtype ligands. Its subtype selective ligand identification rates are significantly better than, and its multi-subtype ligand identification rates are comparable to the best rates of the previously used methods. Our method produced low false-hit rates in screening 13.56 M PubChem, 168,016 MDDR and 657,736 ChEMBLdb compounds. Molecular features important for subtype selectivity were extracted by using the recursive feature elimination feature selection method. These features are consistent with literature-reported features. Our method showed similar performance in searching estrogen receptor subtype selective ligands. Our study demonstrated the usefulness of the two-step target binding and selectivity screening method in searching subtype selective ligands from large compound libraries. PMID:22720033

  13. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3K?-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  14. Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway.

    PubMed

    Fu, Ying-Zi; Yan, Yuan-Yuan; He, Miao; Xiao, Qing-Huan; Yao, Wei-Fan; Zhao, Lin; Wu, Hui-Zhe; Yu, Zhao-Jin; Zhou, Ming-Yi; Lv, Mu-Tian; Zhang, Shan-Shan; Chen, Jian-Jun; Wei, Min-Jie

    2016-02-01

    Breast cancer stem cells (BCSCs) are believed to be responsible for tumor chemoresistance, recurrence, and metastasis formation. Salinomycin (SAL), a carboxylic polyether ionophore, has been reported to act as a selective breast CSC inhibitor. However, the molecular mechanisms underlying SAL-induced cytotoxicity on BCSCs remain unclear. The Hedgehog (Hh) signaling pathway plays an important role in CSC maintenance and carcinogenesis. Here, we investigated whether SAL induces cytotoxicity on BCSCs through targeting Hh pathway. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain breast CSC-enriched MCF-7 mammospheres (MCF-7 MS). MCF-7 MS cells possessed typical BCSC properties, such as CD44+CD24-/low phenotype, high expression of OCT4 (a stem cell marker), increased colony-forming ability, strong migration and invasion capabilities, differentiation potential, and strong tumorigenicity in xenografted mice. SAL exhibited selective cytotoxicity to MCF-7 MS cells relative to MCF-7 cells. The Hh pathway was highly activated in BCSC-enriched MCF-7 MS cells and SAL inhibited Hh signaling activation by downregulating the expression of critical components of the Hh pathway such as PTCH, SMO, Gli1, and Gli2, and subsequently repressing the expression of their essential downstream targets including C-myc, Bcl-2, and Snail (but not cyclin D1). Conversely, Shh-induced Hh signaling activation could largely reverse SAL-mediated inhibitory effects. These findings suggest that SAL-induced selective cytotoxicity against MCF-7 MS cells is associated with the inhibition of Hh signaling activation and the expression of downstream targets and the Hh pathway is an important player and a possible drug target in the pathogenesis of BCSCs. PMID:26718029

  15. Characterization of parasite-specific indels and their proposed relevance for selective anthelminthic drug targeting.

    PubMed

    Wang, Qi; Heizer, Esley; Rosa, Bruce A; Wildman, Scott A; Janetka, James W; Mitreva, Makedonka

    2016-04-01

    Insertions and deletions (indels) are important sequence variants that are considered as phylogenetic markers that reflect evolutionary adaptations in different species. In an effort to systematically study indels specific to the phylum Nematoda and their structural impact on the proteins bearing them, we examined over 340,000 polypeptides from 21 nematode species spanning the phylum, compared them to non-nematodes and identified indels unique to nematode proteins in more than 3000 protein families. Examination of the amino acid composition revealed uneven usage of amino acids for insertions and deletions. The amino acid composition and cost, along with the secondary structure constitution of the indels, were analyzed in the context of their biological pathway associations. Species-specific indels could enable indel-based targeting for drug design in pathogens/parasites. Therefore, we screened the spatial locations of the indels in the parasite's protein 3D structures, determined the location of the indel and identified potential unique drug targeting sites. These indels could be confirmed by RNA-Seq data. Examples are presented illustrating the close proximity of some indels to established small-molecule binding pockets that can potentially facilitate selective targeting to the parasites and bypassing their host, thus reducing or eliminating the toxicity of the potential drugs. This study presents an approach for understanding the adaptation of pathogens/parasites at a molecular level, and outlines a strategy to identify such nematode-selective targets that remain essential to the organism. With further experimental characterization and validation, it opens a possible channel for the development of novel treatments with high target specificity, addressing both host toxicity and resistance concerns. PMID:26829384

  16. Target region selection is a critical determinant of community fingerprints generated by 16S pyrosequencing.

    PubMed

    Kumar, Purnima S; Brooker, Michael R; Dowd, Scot E; Camerlengo, Terry

    2011-01-01

    Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1-V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacter and Enterococcus predominated in the community generated by V4-V6 primers, and the most numerous genera in the V7-V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4-V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7-V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1-V3 and V7-V9 primers provided results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities. PMID:21738596

  17. Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus

    NASA Astrophysics Data System (ADS)

    Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

    2012-10-01

    Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

  18. Lysosomal calcium signaling regulates autophagy via calcineurin and TFEB

    PubMed Central

    Medina, Diego L.; Di Paola, Simone; Peluso, Ivana; Armani, Andrea; De Stefani, Diego; Venditti, Rossella; Montefusco, Sandro; Scotto-Rosato, Anna; Prezioso, Carolina; Forrester, Alison; Settembre, Carmine; Wang, Wuyang; Gao, Qiong; Xu, Haoxing; Sandri, Marco; Rizzuto, Rosario; De Matteis, Maria Antonietta; Ballabio, Andrea

    2016-01-01

    The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signaling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release via mucolipin 1 (MCOLN1) activates calcineurin, which binds and de-phosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis via TFEB required MCOLN1-mediated calcineurin activation, linking lysosomal calcium signaling to both calcineurin regulation and autophagy induction. Thus, the lysosome reveals itself as a hub for the signaling pathways that regulate cellular homeostasis. PMID:25720963

  19. Molecular pathologies of and enzyme replacement therapies for lysosomal diseases.

    PubMed

    Sakuraba, Hitoshi; Sawada, Makoto; Matsuzawa, Fumiko; Aikawa, Sei-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Itoh, Kohji

    2006-08-01

    Lysosomal diseases comprise a group of inherited disorders resulting from defects of lysosomal enzymes and their cofactors, and in many of them the nervous system is affected. Recently, enzyme replacement therapy with recombinant lysosomal enzymes has been clinically available for several lysosomal diseases. Such enzyme replacement therapies can improve non-neurological disorders but is not effective for neurological ones. In this review, we discuss the molecular pathologies of lysosomal diseases from the protein structural aspect, current enzyme replacement therapies, and attempts to develop enzyme replacement therapies effective for lysosomal diseases associated with neurological disorders, i.e., production of enzymes, brain-specific delivery and incorporation of lysosomal enzymes into cells. PMID:16918392

  20. In vivo phage display selection of an ovarian cancer targeting peptide for SPECT/CT imaging

    PubMed Central

    Soendergaard, Mette; Newton-Northup, Jessica R; Deutscher, Susan L

    2014-01-01

    The often fatal outcome of ovarian cancer (OC) is related to inadequate detection methods, which may be overcome by development of nuclear imaging agents. Cancer targeting peptides have been identified using in vivo bacteriophage (phage) display technology; however, the majority of these ligands target tumor vasculature. To overcome this problem, a two-tier phage display method was employed to select an ovarian cancer targeting peptide with good pharmacokinetic and imaging properties. A fUSE5 15-amino acid peptide library was screened against xenografted human OC SKOV-3 tumors in mice, which was followed by selection against enriched SKOV-3 cells. The selected peptide RSLWSDFYASASRGP (J18) was synthesized with a GSG-spacer and a 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) chelator and radiolabeled with 111In. SKOV-3 xenografted mice were used to evaluate the biodistribution and single photon emission computed tomography (SPECT) imaging capabilities of the radiolabeled peptide. Competitive binding experiments using 111In-DOTA-GSG-J18 indicated that the peptide displayed a half maximal inhibitory concentration (IC50) value of 10.5 1.1 ?M. Biodistribution studies revealed that tumor uptake was 1.63 0.68, 0.60 0.32, 0.31 0.12 and 0.10 0.02% injected dose/g at 30 min, 1 h, 2 h and 4 h post-injection of 111In-DOTA-GSG-J18, respectively. SPECT/CT imaging demonstrated good tumor uptake and minimal background binding. This study demonstrated successful utilization of a two-tier phage display selection process to identify an ovarian cancer avid peptide with excellent SPECT/CT imaging capabilities. PMID:25250205

  1. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    NASA Astrophysics Data System (ADS)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  2. WNK4 Enhances the Degradation of NCC through a Sortilin-Mediated Lysosomal Pathway

    PubMed Central

    Zhou, Bo; Zhuang, Jieqiu; Wang, Hua; Cebotaru, Liudmila; Guggino, William B.

    2010-01-01

    WNK kinase is a serine/threonine kinase that plays an important role in electrolyte homeostasis. WNK4 significantly inhibits the surface expression of the sodium chloride co-transporter (NCC) by enhancing the degradation of NCC through a lysosomal pathway, but the mechanisms underlying this trafficking are unknown. Here, we investigated the effect of the lysosomal targeting receptor sortilin on NCC expression and degradation. In Cos-7 cells, we observed that the presence of WNK4 reduced the steady-state amount of NCC by approximately half. Co-transfection with truncated sortilin (a dominant negative mutant) prevented this WNK4-induced reduction in NCC. NCC immunoprecipitated with both wild-type sortilin and, to a lesser extent, truncated sortilin. Immunostaining revealed that WNK4 increased the co-localization of NCC with the lysosomal marker cathepsin D, and NCC co-localized with wild-type sortilin, truncated sortilin, and WNK4 in the perinuclear region. These findings suggest that WNK4 promotes NCC targeting to the lysosome for degradation via a mechanism involving sortilin. PMID:19875813

  3. Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages.

    PubMed

    Aflaki, Elma; Moaven, Nima; Borger, Daniel K; Lopez, Grisel; Westbroek, Wendy; Chae, Jae Jin; Marugan, Juan; Patnaik, Samarjit; Maniwang, Emerson; Gonzalez, Ashley N; Sidransky, Ellen

    2016-02-01

    Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets. PMID:26486234

  4. Siramesine triggers cell death through destabilisation of mitochondria, but not lysosomes.

    PubMed

    ?esen, M Hafner; Repnik, U; Turk, V; Turk, B

    2013-01-01

    A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 ?M. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 ?M siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5-40 ?M); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant ?-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 ?M, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine analogues with high anticancer potential. PMID:24091661

  5. Gold nanorods for target selective SPECT/CT imaging and photothermal therapy in vivo

    PubMed Central

    Jang, Boseung; Park, Seonhwa; Kang, Se Hun; Kim, Joa Kyum; Kim, Seok-Ki; Kim, In-Hoo; Choi, Yongdoo

    2012-01-01

    The development of theranostic agents with high detection sensitivity and antitumor efficacy at low concentration is a challenging task for target selective imaging and therapy of cancers. In this study, folate-conjugated and radioactive-iodine-labeled gold nanorods (GNRs) were designed and synthesized for target selective SPECT/CT imaging and subsequent thermal ablation of folate-receptor-overexpressing cancers. Both (ortho-pyridyl) disulfide-poly(ethylene glycol)-folate and a short peptide, H2N-Tyr-Asn-Asn-Leu-Ala-Cys-OH, were conjugated on the surface of the GNRs through thiol chemistry. The tyrosine in the peptide sequence was introduced for radioactive-iodine labeling through an iodine-tyrosine interaction. The labeling efficiency of radioactive iodine was more than 99%. Radiochemical stability tests on iodine-125-labeled GNRs in human serum showed that 91% of the iodine-125 remained intact on the GNRs after incubation for 24 h. In vitro and in vivo results in this study confirmed the potential utility of folate-conjugated and iodine-125-labeled GNRs as smart theranostic agents. This type of platform may also be useful for the targeted SPECT/CT imaging and photothermal therapy of inflammatory diseases such as atherosclerosis and arthritis, in which folate-receptor-overexpressing macrophages play pivotal roles. PMID:23256055

  6. The contribution of the major planet search surveys to EChO target selection

    NASA Astrophysics Data System (ADS)

    Micela, Giuseppina; Bakos, Gspr .; Lopez-Morales, Mercedes; Maxted, Pierre F. L.; Pagano, Isabella; Sozzetti, Alessandro; Wheatley, Peter J.

    2015-12-01

    The EChO core science will be based on a three tier survey, each with increasing sensitivity, in order to study the population of exo-planets from super-Earths to Jupiter-like planets, in the very hot to temperate zones (temperatures of 300 K - 3000 K) of F to M-type host stars. To achieve a meaningful outcome, an accurate selection of the target sample is needed. In this paper we analyse the targets, suitable for EChO observations, expected to result from a sample of present and forthcoming detection surveys. Present day discovered exoplanets are sufficient to provide a large and diverse sample. However we expect that results from ongoing and planned surveys, aimed at identifying new planets, will increase the sample of smaller planets allowing us to optimize the EChO sample selection. The analysis of the expected yields of a representative set of those surveys both from ground and space shows that they will be able to provide a large number of targets, covering an ample range of planetary and stellar parameters, fitting the EChO capabilities.

  7. PITPs as Targets for Selectively Interfering With Phosphoinositide Signaling in Cells

    PubMed Central

    Nile, Aaron H.; Tripathi, Ashutosh; Yuan, Peihua; Mousley, Carl J.; Suresh, Sundari; Wallace, Iain Michael; Shah, Sweety D.; Pohlhaus, Denise Teotico; Temple, Brenda; Nislow, Corey; Giaever, Guri; Tropsha, Alexander; Davis, Ronald W.; St Onge, Robert P.; Bankaitis, Vytas A.

    2013-01-01

    Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies. PMID:24292071

  8. Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

    PubMed

    Abdul-Hay, Samer O; Bannister, Thomas D; Wang, Hui; Cameron, Michael D; Caulfield, Thomas R; Masson, Amandine; Bertrand, Juliette; Howard, Erin A; McGuire, Michael P; Crisafulli, Umberto; Rosenberry, Terrone R; Topper, Caitlyn L; Thompson, Caroline R; Schrer, Stephan C; Madoux, Franck; Hodder, Peter; Leissring, Malcolm A

    2015-12-18

    Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 ?M, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets. PMID:26398879

  9. Newborn screening for lysosomal storage disorders.

    PubMed

    Matern, Dietrich; Gavrilov, Dimitar; Oglesbee, Devin; Raymond, Kimiyo; Rinaldo, Piero; Tortorelli, Silvia

    2015-04-01

    Every newborn in the U.S. is screened for at least 29 disorders, where evidence suggests that early detection is possible and beneficial. With new or improved treatment options and development of high-throughput screening tests, additional conditions have been proposed for inclusion in newborn screening programs. Among those are several lysosomal storage disorders that have been evaluated in limited pilot studies or that are already included in a few national or international newborn screening programs. These conditions include Pompe disease, Niemann-Pick type A/B disease, Fabry disease, Krabbe disease, Mucopolysaccharidoses types I and II, and Gaucher disease. Here, we review the current state of newborn screening for these lysosomal storage disorders. PMID:25891428

  10. Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses.

    PubMed

    Hildebrand, Laura; Seemann, Petra; Kurtz, Andreas; Hecht, Jochen; Contzen, Jrg; Gossen, Manfred; Stachelscheid, Harald

    2015-12-01

    Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA(+) cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSC(TVA+) as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA(+) cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies. PMID:26109426

  11. Combinatorial selection of DNA thioaptamers targeted towards the HA binding domain of human CD44

    PubMed Central

    Somasunderam, Anoma; Thiviyanathan, Varatharasa; Tanaka, Takemi; Li, Xin; Neerathilingam, Muniasamy; Lokesh, Ganesh Lakshmana Rao; Mann, Aman; Peng, Yang; Ferrari, Mauro; Klostergaard, Jim; Gorenstein, David G.

    2010-01-01

    CD44, the primary receptor for hyaluronic acid, plays an important role in tumor growth and metastasis. CD44-hyaluronic acid interactions can be exploited for targeted delivery of anti-cancer agents specifically to cancer cells. Although various splicing variants of CD44 are expressed on the plasma membrane of cancer cells, the hyaluronic acid binding domain (HABD) is highly conserved among the CD44 splicing variants. Using a novel two-step process, we have identified monothiophosphate-modified aptamers (thioaptamers) that specifically bind to the CD44s HABD with high affinities. Binding affinities of the selected thioaptamers for the HABD were in the range of 180295 nM, significantly higher affinity than that of hyaluronic acid (Kd > ?M range). The selected thioaptamers bound to CD44 positive human ovarian cancer cell lines (SKOV3, IGROV, and A2780), but failed to bind CD44 negative NIH3T3 cell line. Our results indicated that thio substitution at specific positions of the DNA phosphate backbone result in specific and high affinity binding of thioaptamers to CD44. The selected thioaptamers will be of great interest for further development as a targeting or imaging agent to deliver therapeutic payloads for cancer tissues. PMID:20843027

  12. Selective vitamin D receptor activation as anti-inflammatory target in chronic kidney disease.

    PubMed

    Donate-Correa, J; Domnguez-Pimentel, V; Mndez-Prez, M L; Muros-de-Fuentes, M; Mora-Fernndez, C; Martn-Nez, E; Cazaa-Prez, V; Navarro-Gonzlez, J F

    2014-01-01

    Paricalcitol, a selective vitamin D receptor (VDR) activator used for treatment of secondary hyperparathyroidism in chronic kidney disease (CKD), has been associated with survival advantages, suggesting that this drug, beyond its ability to suppress parathyroid hormone, may have additional beneficial actions. In this prospective, nonrandomised, open-label, proof-of-concept study, we evaluated the hypothesis that selective vitamin D receptor activation with paricalcitol is an effective target to modulate inflammation in CKD patients. Eight patients with an estimated glomerular filtration rate between 15 and 44?mL/min/1.73?m(2) and an intact parathyroid hormone (PTH) level higher than 110?pg/mL received oral paricalcitol (1? ?g/48 hours) as therapy for secondary hyperparathyroidism. Nine patients matched by age, sex, and stage of CKD, but a PTH level <110?pg/mL, were enrolled as a control group. Our results show that five months of paricalcitol administration were associated with a reduction in serum concentrations of hs-CRP (13.9%, P < 0.01), TNF-? (11.9%, P = 0.01), and IL-6 (7%, P < 0.05), with a nonsignificant increase of IL-10 by 16%. In addition, mRNA expression levels of the TNF? and IL-6 genes in peripheral blood mononuclear cells decreased significantly by 30.8% (P = 0.01) and 35.4% (P = 0.01), respectively. In conclusion, selective VDR activation is an effective target to modulate inflammation in CKD. PMID:24511210

  13. Lysosomal Sequestration Determines Intracellular Imatinib Levels.

    PubMed

    Burger, Herman; den Dekker, Alexander T; Segeletz, Sandra; Boersma, Antonius W M; de Bruijn, Peter; Debiec-Rychter, Maria; Taguchi, Takahiro; Sleijfer, Stefan; Sparreboom, Alex; Mathijssen, Ron H J; Wiemer, Erik A C

    2015-09-01

    The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (organic cation transporter 1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here, we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses, and apoptosis assays were performed to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562 and SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time-, dose-, temperature-, and energy-dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH4Cl, and the vacuolar ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Costaining experiments with LysoTracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib-mediated cell cycle arrest but significantly increased apoptosis in imatinib-sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression. PMID:26108972

  14. LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation

    PubMed Central

    Milkereit, Ruth; Persaud, Avinash; Vanoaica, Liviu; Guetg, Adriano; Verrey, Francois; Rotin, Daniela

    2015-01-01

    Mammalian target of rapamycin 1 (mTORC1), a master regulator of cellular growth, is activated downstream of growth factors, energy signalling and intracellular essential amino acids (EAAs) such as Leu. mTORC1 activation occurs at the lysosomal membrane, and involves V-ATPase stimulation by intra-lysosomal EAA (inside-out activation), leading to activation of the Ragulator, RagA/B-GTP and mTORC1 via Rheb-GTP. How Leu enters the lysosomes is unknown. Here we identified the lysosomal protein LAPTM4b as a binding partner for the Leu transporter, LAT1-4F2hc (SLC7A5-SLAC3A2). We show that LAPTM4b recruits LAT1-4F2hc to lysosomes, leading to uptake of Leu into lysosomes, and is required for mTORC1 activation via V-ATPase following EAA or Leu stimulation. These results demonstrate a functional Leu transporter at the lysosome, and help explain the inside-out lysosomal activation of mTORC1 by Leu/EAA. PMID:25998567

  15. Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes.

    PubMed

    Singh, Pallavi; Foley, Steven L; Nayak, Rajesh; Kwon, Young Min

    2012-01-01

    Rapid development of next generation sequencing (NGS) technologies in recent years has made whole genome sequencing of bacterial genomes widely accessible. However, it is often unnecessary or not feasible to sequence the whole genome for most applications of genetic analyses in bacteria. Selectively capturing defined genomic regions followed by NGS analysis could be a promising approach for high-resolution molecular typing of a large set of strains. In this study, we describe a novel and straightforward PCR-based target-capturing method, hairpin-primed multiplex amplification (HPMA), which allows for simultaneous amplification of numerous target genes. To test the feasibility of NGS-based strain typing using HPMA, 20 target gene sequences were simultaneously amplified with barcode tagging in each of 41 Salmonella strains. The amplicons were then pooled and analyzed by 454 pyrosequencing. Analysis of the sequence data, as an extension of multilocus sequence typing (MLST), demonstrated the utility and potential of this novel typing method, MLST-seq, as a high-resolution strain typing method. With the rapidly increasing sequencing capacity of NGS, MLST-seq or its variations using different target enrichment methods can be expected to become a high-resolution typing method in the near future for high-throughput analysis of a large collection of bacterial strains. PMID:22108494

  16. Host Factors in Retroviral Integration and the Selection of Integration Target Sites.

    PubMed

    Craigie, Robert; Bushman, Frederic D

    2014-12-01

    In order to replicate, a retrovirus must integrate a DNA copy of the viral RNA genome into a chromosome of the host cell. The study of retroviral integration has advanced considerably in the past few years. Here we focus on host factor interactions and the linked area of integration targeting. Genome-wide screens for cellular factors affecting HIV replication have identified a series of host cell proteins that may mediate subcellular trafficking for preintegration complexes, nuclear import, and integration target site selection. The cell transcriptional co-activator protein LEDGF/p75 has been identified as a tethering factor important for HIV integration, and recently, BET proteins (Brd2, 4, and 4) have been identified as tethering factors for the gammaretroviruses. A new class of HIV inhibitors has been developed targeting the HIV-1 IN-LEDGF binding site, though surprisingly these inhibitors appear to block assembly late during replication and do not act at the integration step. Going forward, genome-wide studies of HIV-host interactions offer many new starting points to investigate HIV replication and identify potential new inhibitor targets. PMID:26104434

  17. MuB gives a new twist to target DNA selection

    PubMed Central

    Dramićanin, Marija; Ramón-Maiques, Santiago

    2013-01-01

    Transposition target immunity is a phenomenon observed in some DNA transposons that are able to distinguish the host chromosome from their own DNA sequence, thus avoiding self-destructive insertions. The first molecular insight into target selection and immunity mechanisms came from the study of phage Mu transposition, which uses the protein MuB as a barrier to self-insertion. MuB is an ATP-dependent non-specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. However, a detailed mechanistic understanding of MuB functioning was hindered by the poor solubility of the MuB-ATP complexes. Here we comment on the recent discovery that MuB is an AAA+ ATPase that upon ATP binding assembles into helical filaments that coat the DNA. Remarkably, the helical parameters of the MuB filament do not match those of the bound DNA. This intriguing mismatch symmetry led us to propose a model on how MuB targets DNA for transposition, favoring DNA bending and recognition by the transposase at the filament edge. We also speculate on a different protective role of MuB during immunity, where filament stickiness could favor the condensation of the DNA into a compact state that occludes it from the transposase. PMID:24478936

  18. Selection of Potential Pharmacological Targets in ALS Based on Whole- Genome Expression Profiling.

    PubMed

    Morello, Giovanna; Conforti, Francesca Luisa; Parenti, Rosalba; D'Agata, Velia; Cavallaro, Sebastiano

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal disease caused by the gradual degeneration and death of upper and lower motor neurons. Despite continue efforts, the etiology and pathogenesis of ALS are not well understood yet. The lack of knowledge about molecular and cellular players involved in the neurodegenerative progression of ALS hinders effective therapy development. Several genomicbased studies have been conducted to identify genetic contributors to sporadic ALS (SALS) and new potential pharmacological targets, but these have resulted in short and non-overlapping lists of candidates. In the last few years, our research group has developed the largest whole-genome expression profile database of SALS human samples. We have identified several genes deregulated in the motor cortex of SALS patients and analyzed the role of these genes within deregulated pathways, providing a full molecular portrait of ALS pathogenesis. Some of deregulated genes encode for proteins that are direct or indirect targets of experimental or therapeutic drugs already applied to unrelated diseases. In this review, we focus on the potential role of candidate targets in ALS pathophysiology, highlighting their possible contribution to ALS therapy. The rational selection of the most promising drug targets and related modulatory drugs may provide a starting point for their preclinical or clinical validation and, hopefully, the development of more effective treatments for ALS patients. PMID:25850769

  19. Acute Targeting of General Transcription Factor IIB Restricts Cardiac Hypertrophy via Selective Inhibition of Gene Transcription

    PubMed Central

    Sayed, Danish; Yang, Zhi; He, Minzhen; Pfleger, Jessica M.; Abdellatif, Maha

    2014-01-01

    Background We previously reported that specialized and housekeeping genes are differentially regulated via de novo recruitment and pause-release of RNA polymerase II (pol II), respectively, during cardiac hypertrophy. However, the significance of this finding remains to be examined. Therefore, the purpose of this study was to determine the mechanisms that differentially regulate these gene groups and exploit them for therapeutic targeting. Methods and Results Here we show that general transcription factor IIB (TFIIB) and cyclin-dependent kinase 9 are upregulated during hypertrophy, both targeted by miR-1, and play preferential roles in regulating those two groups of genes. Chromatin immunoprecipitation-sequencing reveals that TFIIB is constitutively bound to all paused, housekeeping, promoters, whereas, de novo recruitment of TFIIB and pol II is required for specialized genes that are induced during hypertrophy. We exploited this dichotomy to acutely inhibit induction of the latter set, which encompasses cardiomyopathy, immune reaction, and extracellular matrix genes, using locked nucleic acid (LNA)-modified antisense TFIIB oligonucleotide treatment. This resulted in suppression of all specialized genes, while sparing the housekeeping ones, and, thus, attenuated pathological hypertrophy. Conclusions The data for the first time reveal distinct general transcription factor IIB dynamics that regulate specialized vs. housekeeping genes during cardiac hypertrophy. Thus, by acutely targeting TFIIB we were able to selectively inhibit the former set of genes and ameliorate pressure overload hypertrophy. We also demonstrate the feasibility of acutely and reversibly targeting cardiac mRNA for therapeutic purposes using LNA-modified antisense oligonucleotides. PMID:25398966

  20. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  1. Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma.

    PubMed

    Bogen, Dominik; Wei, Jun S; Azorsa, David O; Ormanoglu, Pinar; Buehler, Eugen; Guha, Rajarshi; Keller, Jonathan M; Mathews Griner, Lesley A; Ferrer, Marc; Song, Young K; Liao, Hongling; Mendoza, Arnulfo; Gryder, Berkley E; Sindri, Sivasish; He, Jianbin; Wen, Xinyu; Zhang, Shile; Shern, John F; Yohe, Marielle E; Taschner-Mandl, Sabine; Shohet, Jason M; Thomas, Craig J; Martin, Scott E; Ambros, Peter F; Khan, Javed

    2015-11-01

    Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. PMID:26497213

  2. Intracellular sphingosine releases calcium from lysosomes

    PubMed Central

    Hglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  3. Newborn Screening for Lysosomal Storage Diseases

    PubMed Central

    Gelb, Michael H.; Scott, C. Ronald; Turecek, Frantisek

    2015-01-01

    BACKGROUND There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are critical because of the large number of potential false positives. CONTENT This review summarizes 3 screening methods: (a) direct assay of enzymatic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. SUMMARY Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymatic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable number of screen positives that can be further evaluated with second-tier methods. PMID:25477536

  4. Selection of Nearby Star Targets for the Subaru Strategic Exploration of Exoplanets and Disks

    NASA Astrophysics Data System (ADS)

    Kandori, R.; Tamura, M.; Morino, J.; Ishii, M.; Suzuki, R.; Hashimoto, J.; Kusakabe, N.; Narita, N.; Sato, B.; Yamada, T.; Enya, K.; Goto, M.; Carson, J.; Thalmann, C.; McElwain, M.; Moro-Martin, A.; Knapp, J.; Turner, E. L.

    2009-08-01

    SEEDS (the Subaru Strategic Exploration of Exoplanets and Disks with Hi-CIAO/AO188) is a strategic five-year campaign of direct imaging surveys of exoplanets/disks using the Subaru telescope equipped with the new adaptive optics system AO188 and our new high-contrast instrument, HiCIAO. The goals of the survey are to address the following key issues in exoplanet/disk sciences: (1) the detection and census of exoplanets; (2) the evolution of protoplanetary and debris disks; and (3) the link between exoplanets and disks. Targets prepared for the SEEDS exoplanet searches are in four categories, including nearby stars. We present our scientific motivations and current status of the SEEDS target selection in the nearby stars category.

  5. Selective targeting of green fluorescent nanodiamond conjugates to mitochondria in HeLa cells.

    PubMed

    Mkandawire, Msaukiranji; Pohl, Andrea; Gubarevich, Tatiana; Lapina, Victoria; Appelhans, Dietmar; Rdel, Gerhard; Pompe, Wolfgang; Schreiber, Jrgen; Opitz, Jrg

    2009-10-01

    Fluorescent cellular biomarkers play a prominent role in biosciences. Most of the available biomarkers have some drawbacks due to either physical and optical or cytotoxic properties. In view of this, we investigated the potential of green fluorescent nanodiamonds as biomarkers in living cells. Nanodiamonds were functionalized by attaching antibodies that target intracellular structures such as actin filaments and mitochondria. Then, the nanodiamond conjugates were transfected into HeLa cells. Transfections were mediated by 4(th)-generation dendrimers, cationic liposomes and protamine sulfate. Using fluorescence microscopy, we confirmed successful transfections of the nanodiamonds into HeLa cells. Nanodiamond fluorescence could be easily differentiated from cellular autofluorescence. Furthermore, nanodiamonds could be targeted selectively to intracellular structures. Therefore, nanodiamonds are a promising tool for intracellular assays. PMID:19504515

  6. Candidate Targets of Balancing Selection in the Genome of Staphylococcus aureus

    PubMed Central

    Thomas, Jonathan C.; Godfrey, Paul A.; Feldgarden, Michael; Robinson, D. Ashley

    2012-01-01

    Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajima's D (>2.03) and ?/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen. PMID:22114360

  7. Selection of target mutation in rat gastrointestinal tract E. coli by minute dosage of enrofloxacin

    PubMed Central

    Lin, Dachuan; Chen, Kaichao; Li, Ruichao; Liu, Lizhang; Guo, Jiubiao; Yao, Wen; Chen, Sheng

    2014-01-01

    It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria in vitro. Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry. PMID:25237308

  8. Margin selection to compensate for loss of target dose coverage due to target motion during external-beam radiation therapy of the lung.

    PubMed

    Foster, W Kyle; Osei, Ernest; Barnett, Rob

    2015-01-01

    The aim of this study is to provide guidelines for the selection of external-beam radiation therapy target margins to compensate for target motion in the lung during treatment planning. A convolution model was employed to predict the effect of target motion on the delivered dose distribution. The accuracy of the model was confirmed with radiochromic film measurements in both static and dynamic phantom modes. 502 unique patient breathing traces were recorded and used to simulate the effect of target motion on a dose distribution. A 1D probability density function (PDF) representing the position of the target throughout the breathing cycle was generated from each breathing trace obtained during 4D CT. Changes in the target D95 (the minimum dose received by 95% of the treatment target) due to target motion were analyzed and shown to correlate with the standard deviation of the PDF. Furthermore, the amount of target D95 recovered per millimeter of increased field width was also shown to correlate with the standard deviation of the PDF. The sensitivity of changes in dose coverage with respect to target size was also determined. Margin selection recommendations that can be used to compensate for loss of target D95 were generated based on the simulation results. These results are discussed in the context of clinical plans. We conclude that, for PDF standard deviations less than 0.4 cm with target sizes greater than 5 cm, little or no additional margins are required. Targets which are smaller than 5 cm with PDF standard deviations larger than 0.4 cm are most susceptible to loss of coverage. The largest additional required margin in this study was determined to be 8 mm. PMID:25679166

  9. Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

    PubMed Central

    Lpez Sanjurjo, Cristina I.; Tovey, Stephen C.; Taylor, Colin W.

    2014-01-01

    Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways. PMID:25337829

  10. Target selection and mass estimation for manned NEO exploration using a baseline mission design

    NASA Astrophysics Data System (ADS)

    Boden, Ralf C.; Hein, Andreas M.; Kawaguchi, Junichiro

    2015-06-01

    In recent years Near-Earth Objects (NEOs) have received an increased amount of interest as a target for human exploration. NEOs offer scientifically interesting targets, and at the same time function as a stepping stone for achieving future Mars missions. The aim of this research is to identify promising targets from the large number of known NEOs that qualify for a manned sample-return mission with a maximum duration of one year. By developing a baseline mission design and a mass estimation model, mission opportunities are evaluated based on on-orbit mass requirements, safety considerations, and the properties of the potential targets. A selection of promising NEOs is presented and the effects of mission requirements and restrictions are discussed. Regarding safety aspects, the use of free-return trajectories provides the lowest on-orbit mass, when compared to an alternative design that uses system redundancies to ensure return of the spacecraft to Earth. It is discovered that, although a number of targets are accessible within the analysed time frame, no NEO offers both easy access and high incentive for its exploration. Under the discussed aspects a first human exploration mission going beyond the vicinity of Earth will require a trade off between targets that provide easy access and those that are of scientific interest. This lack of optimal mission opportunities can be seen in the small number of only 4 NEOs that meet all requirements for a sample-return mission and remain below an on-orbit mass of 500 metric Tons (mT). All of them require a mass between 315 and 492 mT. Even less ideal, smaller asteroids that are better accessible require an on-orbit mass that exceeds the launch capability of future heavy lift vehicles (HLV) such as SLS by at least 30 mT. These mass requirements show that additional efforts are necessary to increase the number of available targets and reduce on-orbit mass requirements through advanced mission architectures. The need for on-orbit assembly also becomes apparent, as availability of a HLV alone does not provide sufficient payload capabilities for any manned mission targeting NEOs.

  11. Aptamer-CaCO3 nanostructures: A facile, pH-responsive, specific platform for targeted anticancer theranostics

    PubMed Central

    Zhou, Cuisong; Chen, Tao; Wu, Cuichen; Zhu, Guizhi; Qiu, Liping; Cui, Cheng; Hou, Weijia; Tan, Weihong

    2014-01-01

    Application of cancer theranostics depends on the development of multifunctional nanostructure platforms for accurate cell targeting and controlled drug release, imaging and therapy. In this work, a comprehensive, easily fabricated anticancer theranostic platform with high drug-loading capacity, termed aptamer-functionalized calcium carbonate (CaCO3) nanostructure (apt-CCN), is reported. Flow cytometry and confocal fluorescence microscopy studies demonstrated that apt-CCNs can specifically bind to target cancer cells, but not to control cells, and that they possess highly efficient internalization to target cancer cells. This smart nanostructure selectively reaches the lysosomes through receptor-mediated endocytosis and is responsive to the relatively low lysosome pH (4.55.5), facilitating the release of doxorubicin. The apt-CCN platform offers targeted and efficient drug transport, as well as target-specific delivery of imaging agents for cancer diagnosis and therapy. PMID:25377905

  12. Aptamer CaCO3 nanostructures: a facile, pH-responsive, specific platform for targeted anticancer theranostics.

    PubMed

    Zhou, Cuisong; Chen, Tao; Wu, Cuichen; Zhu, Guizhi; Qiu, Liping; Cui, Cheng; Hou, Weijia; Tan, Weihong

    2015-01-01

    The application of cancer theranostics depends on the development of multifunctional nanostructured platforms for accurate cell targeting and controlled drug release, imaging, and therapy. Herein, a comprehensive, easily fabricated anticancer theranostic platform with a high drug-loading capacity, termed an aptamer-functionalized calcium carbonate (CaCO3 ) nanostructure (apt-CCN), is reported. Flow cytometry and confocal fluorescence microscopy studies demonstrate that apt-CCNs can specifically bind to target cancer cells, but not to control cells, and that they possess highly efficient internalization to target cancer cells. This smart nanostructure selectively reaches the lysosomes through receptor-mediated endocytosis and is responsive to the relatively low lysosome pH (4.5-5.5), which facilitates the release of doxorubicin. The apt-CCN platform offers targeted and efficient drug transport, as well as target-specific delivery of imaging agents for cancer diagnosis and therapy. PMID:25377905

  13. Sequence-based Protein-Protein Interaction Prediction Optimized for Target Selection in Biological Experiments.

    PubMed

    Ye, Jiankuan; Kulikowski, Casimir; Muchnik, Ilya

    2005-01-01

    A set of protein pairs predicted to be interacting with high ratio of true positive is valuable for target selection in experiments like protein structure determination. Our goal in this paper is to investigate the problem of finding such a set of protein pairs in an organism by machine learning methods. Yeast genome was taken for this study and support vector machine was adopted as the classification model. Domain information of each protein was extracted and transformed into features of a protein pair. We specifically analyzed the effect of negative sample selection based on different principles. We also evaluated the feasibility to adjust the intercept parameter of a trained SVM model to improve the ratio of predicted true positive. Our result shows that the approximate 1:3 ratio of positive samples to negative ones in the testing data can be significantly improved to 2:1 of the positive to negative in the predicted data. PMID:17282156

  14. The gaia survey contribution to EChO target selection and characterization

    NASA Astrophysics Data System (ADS)

    Sozzetti, Alessandro; Damasso, Mario

    2015-12-01

    The scientific output of the proposed EChO mission (in terms of spectroscopic characterization of the atmospheres of transiting extrasolar planets) will be maximized by a careful selection of targets and by a detailed characterization of the main physical parameters (such as masses and radii) of both the planets and their stellar hosts. To achieve this aim, the availability of high-quality data from other space-borne and ground-based programs will play a crucial role. Here we identify and discuss the elements of the Gaia catalogue that will be of utmost relevance for the selection and characterization of transiting planet systems to be observed by the proposed EChO mission.

  15. The Role of Microscopy in Understanding Atherosclerotic Lysosomal Lipid Metabolism

    NASA Astrophysics Data System (ADS)

    Gray Jerome, W.; Yancey, Patricia G.

    2003-02-01

    Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

  16. The influence of synesthesia on eye movements: no synesthetic pop-out in an oculomotor target selection task.

    PubMed

    Nijboer, Tanja C W; Satris, Gabriela; Van der Stigchel, Stefan

    2011-12-01

    Recent research on grapheme-colour synesthesia has focused on whether visual attention is necessary to induce a synesthetic percept. The current study investigated the influence of synesthesia on overt visual attention during an oculomotor target selection task. Chromatic and achromatic stimuli were presented with one target among distractors (e.g. a '2' (target) among multiple '5's (distractors)). Participants executed an eye movement to the target. Synesthetes and controls showed a comparable target selection performance across conditions and a 'pop-out effect' was only seen in the chromatic condition. As a pop-out effect was absent for the synesthetes in the achromatic condition, a synesthetic element appears not to elicit a synesthetic colour, even when it is the target. The synesthetic percepts are not pre-attentively available to distinguish the synesthetic target from synesthetic distractors when elements are presented in the periphery. Synesthesia appears to require full recognition to bind form and colour. PMID:21531581

  17. Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene

    SciTech Connect

    Carrer, H.; Maliga, P.

    1995-08-01

    To determine whether targeted DNA insertion into the tobacco plastid genome can be obtained without physical linkage to a selectable marker gene, we carried out biolistic transformation of chloroplasts in tobacco leaf segments with a 1:1 mix of two independently targeted antibiotic resistance genes. Plastid transformants were selected by spectinomycin resistance due to expression of an integrated aadA gene. Integration of the unselected kanamycin resistance (kan) gene into the same plastid genome was established by Southern probing in {approx}20% of the spectinomycin-selected clones. Efficient cotransformation will facilitate targeted plastid genome modification without physical linkage to a marker gene. 26 refs., 5 figs., 1 tab.

  18. The SDSS-IV Extended Baryon Oscillation Spectroscopic Survey: Quasar Target Selection

    NASA Astrophysics Data System (ADS)

    Myers, Adam D.; Palanque-Delabrouille, Nathalie; Prakash, Abhishek; Pâris, Isabelle; Yeche, Christophe; Dawson, Kyle S.; Bovy, Jo; Lang, Dustin; Schlegel, David J.; Newman, Jeffrey A.; Petitjean, Patrick; Kneib, Jean-Paul; Laurent, Pierre; Percival, Will J.; Ross, Ashley J.; Seo, Hee-Jong; Tinker, Jeremy L.; Armengaud, Eric; Brownstein, Joel; Burtin, Etienne; Cai, Zheng; Comparat, Johan; Kasliwal, Mansi; Kulkarni, Shrinivas R.; Laher, Russ; Levitan, David; McBride, Cameron K.; McGreer, Ian D.; Miller, Adam A.; Nugent, Peter; Ofek, Eran; Rossi, Graziano; Ruan, John; Schneider, Donald P.; Sesar, Branimir; Streblyanska, Alina; Surace, Jason

    2015-12-01

    As part of the Sloan Digital Sky Survey (SDSS) IV the extended Baryon Oscillation Spectroscopic Survey (eBOSS) will improve measurements of the cosmological distance scale by applying the Baryon Acoustic Oscillation (BAO) method to quasar samples. eBOSS will adopt two approaches to target quasars over 7500 deg2. First, a “CORE” quasar sample will combine the optical selection in ugriz using a likelihood-based routine called XDQSOz, with a mid-IR-optical color cut. eBOSS CORE selection (to g < 22 or r < 22) should return ˜70 deg-2 quasars at redshifts 0.9 < z < 2.2 and ˜7 deg-2z > 2.1 quasars. Second, a selection based on variability in multi-epoch imaging from the Palomar Transient Factory should recover an additional ˜3-4 deg-2z > 2.1 quasars to g < 22.5. A linear model of how imaging systematics affect target density recovers the angular distribution of eBOSS CORE quasars over 96.7% (76.7%) of the SDSS north (south) Galactic Cap area. The eBOSS CORE quasar sample should thus be sufficiently dense and homogeneous over 0.9 < z < 2.2 to yield the first few-percent-level BAO constraint near \\bar{z}˜ 1.5.eBOSS quasars at z > 2.1 will be used to improve BAO measurements in the Lyα Forest. Beyond its key cosmological goals, eBOSS should be the next-generation quasar survey, comprising >500,000 new quasars and >500,000 uniformly selected spectroscopically confirmed 0.9 < z < 2.2 quasars. At the conclusion of eBOSS, the SDSS will have provided unique spectra for more than 800,000 quasars.

  19. Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA).

    PubMed

    Barinka, Cyril; Ptacek, Jakub; Richter, Antonia; Novakova, Zora; Morath, Volker; Skerra, Arne

    2016-03-01

    Although prostate carcinoma (PCa) is by far the most commonly diagnosed neoplasia in men, corresponding diagnostic and therapeutic modalities have limited efficacy at present. Anticalins comprise a novel class of binding proteins based on a non-immunoglobulin scaffold that can be engineered to specifically address molecular targets of interest. Here we report the selection and characterization of Anticalins that recognize human prostate-specific membrane antigen (PSMA), a membrane-tethered metallopeptidase constituting a disease-related target for imaging and therapy of PCa as well as solid malignancies in general. We used a randomized lipocalin library based on the human lipocalin 2 (Lcn2) scaffold together with phage display and ELISA screening to select PSMA-specific variants. Five Anticalin candidates from the original panning were expressed in Escherichia coli as soluble monomeric proteins, revealing affinities toward PSMA down to the low nanomolar range. Binding characteristics of the most promising candidate were further improved via affinity maturation by applying error-prone PCR followed by selection via phage display as well as bacterial surface display under more stringent conditions. In BIAcore measurements, the dissociation constant of the best Anticalin was determined as ∼500 pM, with a substantially improved dissociation rate compared with the first-generation candidate. Finally, immunofluorescence microscopy revealed specific staining of PSMA-positive tumor cell lines while flow cytometric analysis confirmed the ability of the selected Anticalins to detect PSMA on live cells. Taken together, Anticalins resulting from this study offer a viable alternative to antibody-based PSMA binders for biomedical applications, including in vivo imaging of PCa or neovasculature of solid tumors. PMID:26802163

  20. Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing.

    PubMed

    Lee, Eun-Joon; Pei, Lirong; Srivastava, Gyan; Joshi, Trupti; Kushwaha, Garima; Choi, Jeong-Hyeon; Robertson, Keith D; Wang, Xinguo; Colbourne, John K; Zhang, Lu; Schroth, Gary P; Xu, Dong; Zhang, Kun; Shi, Huidong

    2011-10-01

    We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21,408 CGIs and more than 15,946 transcriptional regulatory regions. Of the CpGs analyzed, 77-84% fell on or near capture probe sequences; 69-75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5'-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples. PMID:21785137

  1. The diffusion properties of ion implanted species in selected target materials

    SciTech Connect

    Alton, G.D.; Dellwo, J.; Carter, H.K.; Kormicki, J.; Bartolo, G. di; Batchelder, J.C.; Breitenbach, J.; Chediak, J.A.; Jentoff-Nilsen, K.; Ichikawa, S.

    1995-02-01

    Experiments important to the future success of the Holifield Radioactive Ion Beam Facility (HRIBF) are in progress at the Oak Ridge National Laboratory which are designed to select the most appropriate target material for generating a particular radioactive ion beam (RIB). The 25-MV HHIRF tandem accelerator is used to implant stable complements of interesting radioactive elements into refractory targets mounted in a high-temperature FEBIAD ion source which is {open_quotes}on-line{close_quotes} at the UNISOR facility. The intensity versus time of implanted species, which diffuse from the high-temperature target material ({approximately}1700{degrees}C) and are ionized in the FEBIAD ion source, is used to determine release times for a particular projectile/target material combination. From such release data, diffusion coefficients can be derived by fitting the theoretical results obtained by computational solution of Fick`s second equation to experimental data. The diffusion coefficient can be used subsequently to predict the release properties of the particular element from the same material in other target geometries and at other temperatures, provided that the activation energy is also known. Diffusion coefficients for Cl implanted into and diffused from CeS and Zr{sub 5}Si{sub 3} and As, Br, and Se implanted into and diffused from Zr{sub 5}Ge{sub 3} have been derived from the resulting intensity versus time profiles. Brief descriptions of the experimental apparatus and procedures utilized in the present experiments and plans for future related experiments are presented.

  2. Lysosomal NEU1 deficiency affects Amyloid Precursor Protein levels and amyloid-? secretion via deregulated lysosomal exocytosis

    PubMed Central

    Annunziata, Ida; Patterson, Annette; Helton, Danielle; Hu, Huimin; Moshiach, Simon; Gomero, Elida; Nixon, Ralph; dAzzo, Alessandra

    2013-01-01

    Alzheimers disease (AD) belongs to a category of adult neurodegenerative conditions which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of A?-peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces ?-amyloid plaques. Our findings identify an additional pathway for the secretion of A? and define NEU1 as a potential therapeutic molecule for AD. PMID:24225533

  3. Selection, characterization, and biosensing application of high affinity congener-specific microcystin-targeting aptamers.

    PubMed

    Ng, Andy; Chinnappan, Raja; Eissa, Shimaa; Liu, Hechun; Tlili, Chaker; Zourob, Mohammed

    2012-10-01

    The efficiency of current microcystin detection methods has been hampered by the low detection limits required in drinking water and that routine detection is restricted to a few of the congeners with high degree of undesired cross-reactivity. Here, we report the development of novel microcystin-targeting molecules and their application in microcystin detection. We have selected DNA aptamers from a diverse random library that exhibit high affinity and specificity to microcystin-LR, -YR, and -LA. We obtained aptamers that bind to all chosen congeners with high affinity with K(D) ranging from 28 to 60 nM. More importantly, we also obtained aptamers that are selective among the different congeners, with selectivity from 3-folds difference in binding affinity to total discrimination (K(D) of 50 nM versus nonspecific binding). Electrochemical aptasensors constructed with the selected aptamers were able to achieve sensitive and congener-specific microcystin detection with detection limit as low as 10 pM. PMID:22958101

  4. Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells

    PubMed Central

    Zou, Jianlong; Hu, Bo; Arpag, Sezgi; Yan, Qing; Hamilton, Audra; Zeng, Yuan-Shan; Vanoye, Carlos G.

    2015-01-01

    Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5′-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4−/−) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4−/− cells. The fission defect was associated with a robust increase of intralysosomal Ca2+ in Fig4−/− cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca2+ efflux of lysosomes because the endogenous ligand of lysosomal Ca2+ channel TRPML1 is PI3,5P2 that is deficient in Fig4−/− cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca2+ level and rescued abnormal lysosomal storage in Fig4−/− culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca2+ efflux in Fig4−/− culture cells and Fig4−/− mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency. PMID:25926456

  5. Does Angling Technique Selectively Target Fishes Based on Their Behavioural Type?

    PubMed Central

    Wilson, Alexander D. M.; Brownscombe, Jacob W.; Sullivan, Brittany; Jain-Schlaepfer, Sofia; Cooke, Steven J.

    2015-01-01

    Recently, there has been growing recognition that fish harvesting practices can have important impacts on the phenotypic distributions and diversity of natural populations through a phenomenon known as fisheries-induced evolution. Here we experimentally show that two common recreational angling techniques (active crank baits versus passive soft plastics) differentially target wild largemouth bass (Micropterus salmoides) and rock bass (Ambloplites rupestris) based on variation in their behavioural tendencies. Fish were first angled in the wild using both techniques and then brought back to the laboratory and tested for individual-level differences in common estimates of personality (refuge emergence, flight-initiation-distance, latency-to-recapture and with a net, and general activity) in an in-lake experimental arena. We found that different angling techniques appear to selectively target these species based on their boldness (as characterized by refuge emergence, a standard measure of boldness in fishes) but not other assays of personality. We also observed that body size was independently a significant predictor of personality in both species, though this varied between traits and species. Our results suggest a context-dependency for vulnerability to capture relative to behaviour in these fish species. Ascertaining the selective pressures angling practices exert on natural populations is an important area of fisheries research with significant implications for ecology, evolution, and resource management. PMID:26284779

  6. Training, selection, and robust calibration of retention time models for targeted proteomics.

    PubMed

    Moruz, Luminita; Tomazela, Daniela; Kll, Lukas

    2010-10-01

    Accurate predictions of peptide retention times (RT) in liquid chromatography have many applications in mass spectrometry-based proteomics. Most notably such predictions are used to weed out incorrect peptide-spectrum matches, and to design targeted proteomics experiments. In this study, we describe a RT predictor, ELUDE, which can be employed in both applications. ELUDE's predictions are based on 60 features derived from the peptide's amino acid composition and optimally combined using kernel regression. When sufficient data is available, ELUDE derives a retention time index for the condition at hand making it fully portable to new chromatographic conditions. In cases when little training data is available, as often is the case in targeted proteomics experiments, ELUDE selects and calibrates a model from a library of pretrained predictors. Both model selection and calibration are carried out via robust statistical methods and thus ELUDE can handle situations where the calibration data contains erroneous data points. We benchmarked our method against two state-of-the-art predictors and showed that ELUDE outperforms these methods and tracked up to 34% more peptides in a theoretical SRM method creation experiment. ELUDE is freely available under Apache License from http://per-colator.com. PMID:20735070

  7. Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

    SciTech Connect

    Shi, Tujin; Su, Dian; Liu, Tao; Tang, Keqi; Camp, David G.; Qian, Weijun; Smith, Richard D.

    2012-04-01

    Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the pg/mL to low ng/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in the cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides or their posttranslational modifications (PTMs), as well as advances in MS instrumentation, which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.

  8. Selective targeting of bioengineered platelets to prostate cancer vasculature: new paradigm for therapeutic modalities

    PubMed Central

    Montecinos, Viviana P; Morales, Claudio H; Fischer, Thomas H; Burns, Sarah; San Francisco, Ignacio F; Godoy, Alejandro S; Smith, Gary J

    2015-01-01

    Androgen deprivation therapy (ADT) provides palliation for most patients with advanced prostate cancer (CaP); however, greater than 80% subsequently fail ADT. ADT has been indicated to induce an acute but transient destabilization of the prostate vasculature in animal models and humans. Human re-hydrated lyophilized platelets (hRL-P) were investigated as a prototype for therapeutic agents designed to target selectively the tumour-associated vasculature in CaP. The ability of hRL-P to bind the perturbed endothelial cells was tested using thrombin- and ADP-activated human umbilical vein endothelial cells (HUVEC), as well as primary xenografts of human prostate tissue undergoing acute vascular involution in response to ADT. hRL-P adhered to activated HUVEC in a dose-responsive manner. Systemically administered hRL-P, and hRL-P loaded with super-paramagnetic iron oxide (SPIO) nanoparticles, selectively targeted the ADT-damaged human microvasculature in primary xenografts of human prostate tissue. This study demonstrated that hRL-P pre-loaded with chemo-therapeutics or nanoparticles could provide a new paradigm for therapeutic modalities to prevent the rebound/increase in prostate vasculature after ADT, inhibiting the transition to castration-recurrent growth. PMID:25736582

  9. Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

    PubMed Central

    Shi, Tujin; Su, Dian; Liu, Tao; Tang, Keqi; Camp, David G.; Qian, Wei-Jun; Smith, Richard D.

    2012-01-01

    Selected reaction monitoring (SRM)also known as multiple reaction monitoring (MRM)has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications (PTMs), as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed. PMID:22577010

  10. Selective targeting of bioengineered platelets to prostate cancer vasculature: new paradigm for therapeutic modalities.

    PubMed

    Montecinos, Viviana P; Morales, Claudio H; Fischer, Thomas H; Burns, Sarah; San Francisco, Ignacio F; Godoy, Alejandro S; Smith, Gary J

    2015-07-01

    Androgen deprivation therapy (ADT) provides palliation for most patients with advanced prostate cancer (CaP); however, greater than 80% subsequently fail ADT. ADT has been indicated to induce an acute but transient destabilization of the prostate vasculature in animal models and humans. Human re-hydrated lyophilized platelets (hRL-P) were investigated as a prototype for therapeutic agents designed to target selectively the tumour-associated vasculature in CaP. The ability of hRL-P to bind the perturbed endothelial cells was tested using thrombin- and ADP-activated human umbilical vein endothelial cells (HUVEC), as well as primary xenografts of human prostate tissue undergoing acute vascular involution in response to ADT. hRL-P adhered to activated HUVEC in a dose-responsive manner. Systemically administered hRL-P, and hRL-P loaded with super-paramagnetic iron oxide (SPIO) nanoparticles, selectively targeted the ADT-damaged human microvasculature in primary xenografts of human prostate tissue. This study demonstrated that hRL-P pre-loaded with chemo-therapeutics or nanoparticles could provide a new paradigm for therapeutic modalities to prevent the rebound/increase in prostate vasculature after ADT, inhibiting the transition to castration-recurrent growth. PMID:25736582

  11. Update on the Pfam5000 Strategy for Selection of StructuralGenomics Targets

    SciTech Connect

    Chandonia, John-Marc; Brenner, Steven E.

    2005-06-27

    Structural Genomics is an international effort to determine the three-dimensional shapes of all important biological macromolecules, with a primary focus on proteins. Target proteins should be selected according to a strategy that is medically and biologically relevant, of good financial value, and tractable. In 2003, we presented the ''Pfam5000'' strategy, which involves selecting the 5,000 most important families from the Pfam database as sources for targets. In this update, we show that although both the Pfam database and the number of sequenced genomes have increased in size, the expected benefits of the Pfam5000 strategy have not changed substantially. Solving the structures of proteins from the 5,000 largest Pfam families would allow accurate fold assignment for approximately 65 percent of all prokaryotic proteins (covering 54 percent of residues) and 63 percent of eukaryotic proteins (42 percent of residues). Fewer than 2,300 of the largest families on this list remain to be solved, making the project feasible in the next five years given the expected throughput to be achieved in the production phase of the Protein Structure Initiative.

  12. Getting to the source: selective drug targeting of cancer stem cells.

    PubMed

    Ismail, Fatima; Winkler, David A

    2014-05-01

    Cancers are among the most important and most difficult to treat diseases of the 21st century. Conventional therapies include surgery, immunotherapy, and radiotherapy, as well many forms of drug treatments such as tamoxifen and Gleevec. However, these forms of treatment often do not eradicate the cancer stem cells, only managing to decrease the size of the tumor, allowing the cancer to return. The cancer stem cell hypothesis stipulates that malignancy is maintained through self-renewal of cancer stem cells (CSCs), which generate rapidly dividing progeny that comprise the tumors, and that are largely untouched by conventional therapies. Evidence for the central role of CSCs in many tumors has provided a paradigm shift in the way cancer chemotherapy may be addressed. Recent discoveries regarding the nature of the stem cell niche, and the key signaling pathways involved in stem cell self-renewal and differentiation from regenerative medicine, have provided key information that facilitates selective targeting of CSCs by small-molecule drugs. The growing body of biochemical knowledge on the nature of CSCs, and differences between them and normal adult stem cells essential for maintaining organisms, has augmented the increasing number of small molecules shown to control normal and aberrant stem cells. Here, we review small-molecule approaches to the selective targeting of CSCs. PMID:24760779

  13. Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

    PubMed Central

    Newton, Phillip T; Vuppalapati, Karuna K; Bouderlique, Thibault; Chagin, Andrei S

    2015-01-01

    Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H+-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity. PMID:26259639

  14. Induction of Lysosomal Biogenesis in Atherosclerotic Macrophages Can Rescue Lipid-Induced Lysosomal Dysfunction and Downstream Sequelae

    PubMed Central

    Bhattacharya, Somashubhra; Turner, Jaleisa; Epelman, Slava; Settembre, Carmine; Diwan, Abhinav; Ballabio, Andrea; Razani, Babak

    2014-01-01

    Objective Recent reports of a proatherogenic phenotype in mice with macrophage-specific autophagy deficiency has renewed interest in the role of the autophagy-lysosomal system in atherosclerosis. Lysosomes have the unique ability to process both exogenous material including lipids and autophagy-derived cargo such as dysfunctional proteins/organelles. We aimed to understand the effects of an atherogenic lipid environment on macrophage lysosomes and evaluate novel ways to modulate this system. Approach and Results Utilizing a variety of complementary techniques, we show that oxidized LDL and cholesterol crystals, commonly encountered lipid species in atherosclerosis, lead to profound lysosomal dysfunction in cultured macrophages. Disruptions in lysosomal pH, proteolytic capacity, membrane integrity, and morphology are readily seen. Using flow cytometry, we find that macrophages isolated from atherosclerotic plaques also display features of lysosome dysfunction. We then investigated whether enhancing lysosomal function can be beneficial. TFEB is the only known transcription factor that is a master regulator of lysosomal biogenesis, although its role in macrophages has not been studied. Lysosomal stress induced by chloroquine or atherogenic lipids leads to TFEB nuclear translocation and activation of lysosomal and autophagy genes. TFEB overexpression in macrophages further augments this prodegradative response and rescues several deleterious effects seen with atherogenic lipid loading as evidenced by blunted lysosomal dysfunction, reduced secretion of the proinflammatory cytokine IL-1?, enhanced cholesterol efflux, and decreased polyubiquitinated protein aggregation. Conclusions Taken together, these data demonstrate that lysosomal function is markedly impaired in atherosclerosis and suggest that induction of a lysosomal biogenesis program in macrophages has anti-atherogenic effects. PMID:25060788

  15. Selection of flowing liquid lead target structural materials for accelerator driven transmutation applications

    SciTech Connect

    Park, J.J.; Buksa, J.J.

    1994-08-01

    The beam entry window and container for a liquid lead spallation target will be exposed to high fluxes of protons and neutrons that are both higher in magnitude and energy than have been experienced in proton accelerators and fission reactors, as well as in a corrosive environment. The structural material of the target should have a good compatibility with liquid lead, a sufficient mechanical strength at elevated temperatures, a good performance under an intense irradiation environment, and a low neutron absorption cross section; these factors have been used to rank the applicability of a wide range of materials for structural containment Nb-1Zr has been selected for use as the structural container for the LANL ABC/ATW molten lead target. Corrosion and mass transfer behavior for various candidate structural materials in liquid lead are reviewed, together with the beneficial effects of inhibitors and various coatings to protect substrate against liquid lead corrosion. Mechanical properties of some candidate materials at elevated temperatures and the property changes resulting from 800 MeV proton irradiation are also reviewed.

  16. Characterizing the pocketome of Mycobacterium tuberculosis and application in rationalizing polypharmacological target selection.

    PubMed

    Anand, Praveen; Chandra, Nagasuma

    2014-01-01

    Polypharmacology is beginning to emerge as an important concept in the field of drug discovery. However, there are no established approaches to either select appropriate target sets or design polypharmacological drugs. Here, we propose a structural-proteomics approach that utilizes the structural information of the binding sites at a genome-scale obtained through in-house algorithms to characterize the pocketome, yielding a list of ligands that can participate in various biochemical events in the mycobacterial cell. The pocket-type space is seen to be much larger than the sequence or fold-space, suggesting that variations at the site-level contribute significantly to functional repertoire of the organism. All-pair comparisons of binding sites within Mycobacterium tuberculosis (Mtb), pocket-similarity network construction and clustering result in identification of binding-site sets, each containing a group of similar binding sites, theoretically having a potential to interact with a common set of compounds. A polypharmacology index is formulated to rank targets by incorporating a measure of druggability and similarity to other pockets within the proteome. This study presents a rational approach to identify targets with polypharmacological potential along with possible drugs for repurposing, while simultaneously, obtaining clues on lead compounds for use in new drug-discovery pipelines. PMID:25220818

  17. Scientific objectives and selection of targets for the SMART-1 Infrared Spectrometer (SIR)

    USGS Publications Warehouse

    Basilevsky, A.T.; Keller, H.U.; Nathues, A.; Mall, U.; Hiesinger, H.; Rosiek, M.

    2004-01-01

    The European SMART-1 mission to the Moon, primarily a testbed for innovative technologies, was launched in September 2003 and will reach the Moon in 2005. On board are several scientific instruments, including the point-spectrometer SMART-1 Infrared Spectrometer (SIR). Taking into account the capabilities of the SMART-1 mission and the SIR instrument in particular, as well as the open questions in lunar science, a selection of targets for SIR observations has been compiled. SIR can address at least five topics: (1) Surface/regolith processes; (2) Lunar volcanism; (3) Lunar crust structure; (4) Search for spectral signatures of ices at the lunar poles; and (5) Ground truth and study of geometric effects on the spectral shape. For each topic we will discuss specific observation modes, necessary to achieve our scientific goals. The majority of SIR targets will be observed in the nadir-tracking mode. More than 100 targets, which require off-nadir pointing and off-nadir tracking, are planned. It is expected that results of SIR observations will significantly increase our understanding of the Moon. Since the exact arrival date and the orbital parameters of the SMART-1 spacecraft are not known yet, a more detailed planning of the scientific observations will follow in the near future. ?? 2004 Elsevier Ltd. All rights reserved.

  18. Phosphatidylserine-selective targeting and anticancer effects of SapC-DOPS nanovesicles on brain tumors

    PubMed Central

    Blanco, Víctor M.; Chu, Zhengtao; Vallabhapurapu, Subrahmanya D.; Sulaiman, Mahaboob K.; Kendler, Ady; Rixe, Olivier; Warnick, Ronald E.; Franco, Robert S.; Qi, Xiaoyang

    2014-01-01

    Brain tumors, either primary (e.g., glioblastoma multiforme) or secondary (metastatic), remain among the most intractable and fatal of all cancers. We have shown that nanovesicles consisting of Saposin C (SapC) and dioleylphosphatidylserine (DOPS) are able to effectively target and kill cancer cells both in vitro and in vivo. These actions are a consequence of the affinity of SapC-DOPS for phosphatidylserine, an acidic phospholipid abundantly present in the outer membrane of a variety of tumor cells and tumor-associated vasculature. In this study, we first characterize SapC-DOPS bioavailability and antitumor effects on human glioblastoma xenografts, and confirm SapC-DOPS specificity towards phosphatidylserine by showing that glioblastoma targeting is abrogated after in vivo exposure to lactadherin, which binds phosphatidylserine with high affinity. Second, we demonstrate that SapC-DOPS selectively targets brain metastases-forming cancer cells both in vitro, in co-cultures with human astrocytes, and in vivo, in mouse models of brain metastases derived from human breast or lung cancer cells. Third, we demonstrate that SapC-DOPS nanovesicles have cytotoxic activity against metastatic breast cancer cells in vitro, and prolong the survival of mice harboring brain metastases. Taken together, these results support the potential of SapC-DOPS for the diagnosis and therapy of primary and metastatic brain tumors. PMID:25051370

  19. Selective targeting of G-protein-coupled receptor subtypes with venom peptides.

    PubMed

    Näreoja, K; Näsman, J

    2012-02-01

    The G-protein-coupled receptor (GPCR) family is one of the largest gene superfamilies with approx. 370 members responding to endogenous ligands in humans and a roughly equal amount of receptors sensitive to external stimuli from the surrounding. A number of receptors from this superfamily are well recognized targets for medical treatment of various disease conditions, whereas for many others the potential medical benefit of interference is still obscure. A general problem associated with GPCR research and therapeutics is the insufficient specificity of available ligands to differentiate between closely homologous receptor subtypes. In this context, venom peptides could make a significant contribution to the development of more specific drugs. Venoms from certain animals specialized in biochemical hunting contain a mixture of molecules that are directed towards a variety of membrane proteins. Peptide toxins isolated from these mixtures usually exhibit high specificity for their targets. Muscarinic toxins found from mamba snakes attracted much attention during the 1990s. These are 65-66 amino acid long peptides with a structural three-finger folding similar to the α-neurotoxins and they target the muscarinic acetylcholine receptors in a subtype-selective manner. Recently, several members of the three-finger toxins from mamba snakes as well as conotoxins from marine cone snails have been shown to selectively interact with subtypes of adrenergic receptors. In this review, we will discuss the GPCR-directed peptide toxins found from different venoms and how some of these can be useful in exploring specific roles of receptor subtypes. PMID:21481193

  20. Selective target inactivation rather than global metabolic dormancy causes antibiotic tolerance in uropathogens.

    PubMed

    Goneau, Lee W; Yeoh, Nigel S; MacDonald, Kyle W; Cadieux, Peter A; Burton, Jeremy P; Razvi, Hassan; Reid, Gregor

    2014-01-01

    Persister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed between Staphylococcus saprophyticus and Escherichia coli uropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancy per se cannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenic E. coli isolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure. PMID:24449771

  1. Selective Target Inactivation Rather than Global Metabolic Dormancy Causes Antibiotic Tolerance in Uropathogens

    PubMed Central

    Goneau, Lee W.; Yeoh, Nigel S.; MacDonald, Kyle W.; Cadieux, Peter A.; Burton, Jeremy P.; Razvi, Hassan

    2014-01-01

    Persister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed between Staphylococcus saprophyticus and Escherichia coli uropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancy per se cannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenic E. coli isolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure. PMID:24449771

  2. Intracellular replication of Salmonella within epithelial cells is associated with filamentous structures containing lysosomal membrane glycoproteins.

    PubMed

    Garcia-del Portillo, F; Zwick, M B; Leung, K Y; Finlay, B B

    1993-08-01

    We have examined the targeting of S. typhimurium-containing vacuoles to lysosomes after invasion of cultured HeLa epithelial cells. Our results show that intracellular bacteria colocalize with vacuoles containing lysosomal membrane glycoproteins (LGPs). Both human LGPs, hlamp-1 and hlamp-2, are present in S. typhimurium-containing vacuoles from approximately 2 h postinfection. At later times (4-6 h), long and stable filamentous structures with lysosomal markers appear connected to bacteria-containing vacuoles in infected cells. Viable intracellular bacteria are required for the formation of these structures, which are not detected in uninfected cells or in HeLa epithelial cells infected with another invasive bacteria, Yersinia enterocolitica. Kinetics analysis showed a strict correlation between the appearance of these LGP-rich filaments and the initiation of intracellular bacterial replication. Moreover, these structures are absent in epithelial cells infected with certain S. typhimurium intracellular replication-defective mutants. Additional data confirmed that an intact microtubule network and intravacuolar acidic pH are required to induce the formation of LGP-containing filamentous structures and that these structures are morphologically and functionally different from previously described tubular lysosomes. PMID:8173800

  3. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells.

    PubMed

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrP(Sc)) of the cellular prion protein (PrP(c)). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrP(c) into PrP(Sc). Within neurons, PrP(Sc) accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrP(Sc) accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  4. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells

    PubMed Central

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M.; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrPSc accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  5. Rag GTPases are cardioprotective by regulating lysosomal function.

    PubMed

    Kim, Young Chul; Park, Hyun Woo; Sciarretta, Sebastiano; Mo, Jung-Soon; Jewell, Jenna L; Russell, Ryan C; Wu, Xiaohui; Sadoshima, Junichi; Guan, Kun-Liang

    2014-01-01

    The Rag family proteins are Ras-like small GTPases that have a critical role in amino-acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases, although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised owing to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection. PMID:24980141

  6. Rag GTPases are cardioprotective by regulating lysosomal function

    PubMed Central

    Kim, Young Chul; Mo, Jung-Soon; Jewell, Jenna L.; Russell, Ryan C.; Wu, Xiaohui; Sadoshima, Junichi; Guan, Kun-Liang

    2014-01-01

    The Rag family proteins are Ras-like small GTPases that play a critical role in amino acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here, we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised due to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection. PMID:24980141

  7. Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

    PubMed

    Berg, Russell D; Levitte, Steven; O'Sullivan, Mary P; O'Leary, Seónadh M; Cambier, C J; Cameron, James; Takaki, Kevin K; Moens, Cecilia B; Tobin, David M; Keane, Joseph; Ramakrishnan, Lalita

    2016-03-24

    A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis. PMID:27015311

  8. Recombinogenic properties of Pyrococcus furiosus strain COM1 enable rapid selection of targeted mutants.

    PubMed

    Farkas, Joel; Stirrett, Karen; Lipscomb, Gina L; Nixon, William; Scott, Robert A; Adams, Michael W W; Westpheling, Janet

    2012-07-01

    We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or "pop-out," of the selected marker. We used this method to construct a "markerless" deletion of the trpAB locus in the GLW101 (COM1 ?pyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene. PMID:22544252

  9. Selective inhibitors of fatty acid amide hydrolase relative to neuropathy target esterase and acetylcholinesterase: toxicological implications.

    PubMed

    Quistad, Gary B; Sparks, Susan E; Segall, Yoffi; Nomura, Daniel K; Casida, John E

    2002-02-15

    Fatty acid amide hydrolase (FAAH) plays an important role in nerve function by regulating the action of endocannabinoids (e.g., anandamide) and hydrolyzing a sleep-inducing factor (oleamide). Several organophosphorus pesticides and related compounds are shown in this study to be more potent in vivo inhibitors of mouse brain FAAH than neuropathy target esterase (NTE), raising the question of the potential toxicological relevance of FAAH inhibition. These FAAH-selective compounds include tribufos and (R)-octylbenzodioxaphosphorin oxide with delayed neurotoxic effects in mice and hens plus several organophosphorus pesticides (e.g., fenthion) implicated as delayed neurotoxicants in humans. The search for a highly potent and selective inhibitor for FAAH relative to NTE for use as a toxicological probe culminated in the discovery that octylsulfonyl fluoride inhibits FAAH by 50% at 2 nM in vitro and 0.2 mg/kg in vivo and NTE is at least 100-fold less sensitive in each case. More generally, the studies revealed 12 selective in vitro inhibitors for FAAH (mostly octylsulfonyl and octylphosphonyl derivatives) and 9 for NTE (mostly benzodioxaphosphorin oxides and organophosphorus fluoridates). The overall in vivo findings with 16 compounds indicate the expected association of AChE inhibition with acute or cholinergic syndrome and >70% brain NTE inhibition with delayed neurotoxic action. Surprisingly, 75-99% brain FAAH inhibition does not lead to any overt neurotoxicity or change in behavior (other than potentiation of exogenous anandamide action). Thus, FAAH inhibition in mouse brain does not appear to be a primary target for organophosphorus pesticide-induced neurotoxic action (cholinergic or intermediate syndrome or delayed neurotoxicity). PMID:11884237

  10. Bezielle Selectively Targets Mitochondria of Cancer Cells to Inhibit Glycolysis and OXPHOS

    PubMed Central

    Chen, Vivian; Staub, Richard E.; Fong, Sylvia; Tagliaferri, Mary; Cohen, Isaac; Shtivelman, Emma

    2012-01-01

    Bezielle (BZL101) is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells. PMID:22319564

  11. Target Selection Signals Influence Perceptual Decisions by Modulating the Onset and Rate of Evidence Accumulation.

    PubMed

    Loughnane, Gerard M; Newman, Daniel P; Bellgrove, Mark A; Lalor, Edmund C; Kelly, Simon P; O'Connell, Redmond G

    2016-02-22

    Computational and neurophysiological research has highlighted neural processes that accumulate sensory evidence for perceptual decisions [1]. These processes have been studied in the context of highly simplified perceptual discrimination paradigms in which the physical evidence appears at times and locations that are either entirely predictable or exogenously cued (e.g., by the onset of the stimulus itself). Yet, we are rarely afforded such certainty in everyday life. For example, when driving along a busy motorway, we must continually monitor the movements of surrounding vehicles for events that call for a lane change. In such scenarios, it is unknown which of the continuously present information sources will become relevant or when. Although it is well established that evidence integration provides an effective mechanism for countering the impact of noise [2], the question of how this mechanism is implemented in the face of uncertain evidence onsets has yet to be answered. Here, we show that when monitoring two potential sources of information for evidence occurring unpredictably in both time and space, the human brain employs discrete, early target selection signals that significantly modulate the onset and rate of neural evidence accumulation, and thereby the timing and accuracy of perceptual reports. These selection signals share many of the key characteristics of the N2pc component highlighted in the literature on visual search [3,4] yet are present even in the absence of distractors and under situations of low temporal and spatial uncertainty. These data provide novel insights into how target selection supports decision making in uncertain environments. PMID:26853360

  12. FAMACHA: A potential tool for targeted selective treatment of chronic fasciolosis in sheep.

    PubMed

    Olah, Sophie; van Wyk, Jan A; Wall, Richard; Morgan, Eric R

    2015-09-15

    The liver fluke Fasciola hepatica causes considerable damage to the health, welfare and productivity of ruminants in temperate areas, and its control is challenged by anthelmintic resistance. Targeted selective treatment (TST) is an increasingly established strategy for preserving anthelmintic efficacy in grazing livestock, yet no practical indicators are available to target individuals for treatment against fluke infection. This paper evaluates the FAMACHA() system, a colour chart for the non-invasive detection of anaemia in small ruminants, for this purpose. FAMACHA() scores were collected from 288 sheep prior to slaughter during the winter period, when fluke infections were largely mature, and condemned livers were recovered and adult flukes extracted. Average FAMACHA() score was significantly higher (=paler conjunctivae) in animals whose livers were condemned (3.6, n=62) than in those whose livers were not condemned (2.1). The number of adult flukes recovered ranged from 2 to 485, and was positively correlated with FAMACHA() score (r(2)=0.54, p<0.001). Packed cell volume was correlated negatively with both FAMACHA() score (n=240, r=0.23, p<0.001) and fluke number (r=0.24, p<0.001). Nematode faecal egg count (FEC) did not correlate with FAMACHA() score, and selective treatment of individual sheep with FAMACHA() scores above 2 or 3 would have preserved between 27 and 100% of nematodes in refugia on the basis of FEC, depending on group and the threshold used for treatment. FAMACHA() holds promise as a tool for selective treatment of sheep against adult F. hepatica, in support of refugia-based control of fluke and nematode infections, and further field evaluation is warranted. PMID:26223154

  13. Isolation of pig thyroid lysosomes. Biochemical and morphological characterization.

    PubMed Central

    Alquier, C; Guenin, P; Munari-Silem, Y; Audebet, C; Rousset, B

    1985-01-01

    Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, beta-galactosidase and beta-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells. Images Fig. 4. PMID:3004408

  14. Selection Against Maternal microRNA Target Sites in Maternal Transcripts

    PubMed Central

    Marco, Antonio

    2015-01-01

    In animals, before the zygotic genome is expressed, the egg already contains gene products deposited by the mother. These maternal products are crucial during the initial steps of development. In Drosophila melanogaster, a large number of maternal products are found in the oocyte, some of which are indispensable. Many of these products are RNA molecules, such as gene transcripts and ribosomal RNAs. Recently, microRNAs (small RNA gene regulators) have been detected early during development and are important in these initial steps. The presence of some microRNAs in unfertilized eggs has been reported, but whether they have a functional impact in the egg or early embryo has not being explored. I have extracted and sequenced small RNAs from Drosophila unfertilized eggs. The unfertilized egg is rich in small RNAs and contains multiple microRNA products. Maternal microRNAs often are encoded within the intron of maternal genes, suggesting that many maternal microRNAs are the product of transcriptional hitchhiking. Comparative genomics analyses suggest that maternal transcripts tend to avoid target sites for maternal microRNAs. I also developed a microRNA target mutation model to study the functional impact of polymorphisms at microRNA target sites. The analysis of Drosophila populations suggests that there is selection against maternal microRNA target sites in maternal transcripts. A potential role of the maternal microRNA mir-9c in maternal-to-zygotic transition is also discussed. In conclusion, maternal microRNAs in Drosophila have a functional impact in maternal protein?coding transcripts. PMID:26306531

  15. Insect-selective spider toxins targeting voltage-gated sodium channels.

    PubMed

    Nicholson, Graham M

    2007-03-15

    The voltage-gated sodium (Na(v)) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Na(v) channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Na(v) channel conductance. Magi 2 and Tx4(6-1) slow Na(v) channel inactivation via an interaction with site-3. The delta-palutoxins, and most likely mu-agatoxins and curtatoxins, target site-4. However, their action is complex with the mu-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion beta-toxins, but with both delta-palutoxins and mu-agatoxins slowing Na(v) channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as delta-atracotoxins, are known to target both insect and vertebrate Na(v) channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped. PMID:17223149

  16. Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

    PubMed Central

    Euler, Chad W.; Juncosa, Barbara; Ryan, Patricia A.; Deutsch, Douglas R.; McShan, W. Michael; Fischetti, Vincent A.

    2016-01-01

    Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution. PMID:26756207

  17. Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique.

    PubMed

    Euler, Chad W; Juncosa, Barbara; Ryan, Patricia A; Deutsch, Douglas R; McShan, W Michael; Fischetti, Vincent A

    2016-01-01

    Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution. PMID:26756207

  18. Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat.

    PubMed

    Robb, Ellen L; Gawel, Justyna M; Aksentijević, Dunja; Cochemé, Helena M; Stewart, Tessa S; Shchepinova, Maria M; Qiang, He; Prime, Tracy A; Bright, Thomas P; James, Andrew M; Shattock, Michael J; Senn, Hans M; Hartley, Richard C; Murphy, Michael P

    2015-12-01

    Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo. PMID:26454075

  19. Production impact of a targeted selective treatment system based on liveweight gain in a commercial flock.

    PubMed

    Busin, V; Kenyon, F; Parkin, T; McBean, D; Laing, N; Sargison, N D; Ellis, K

    2014-05-01

    The sustainability of sheep production is hindered by anthelmintic resistance. Options to slow down or prevent resistance have been widely studied but their application in the field is still limited. In this study, the practical application and effect of a targeted selective treatment (TST) approach