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 ?-cyclodextrin (?-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 ?v?3 and ?v?5 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. Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs

    Microsoft Academic Search

    Matthew Bogyo; Steven Verhelst; Valerie Bellingard-Dubouchaud; Sam Toba; Doron Greenbaum

    2000-01-01

    Background: The lysosomal cysteine proteases of the papain family are some of the best studied proteolytic enzymes. Small-molecule inhibitors and fluorogenic substrate mimics have been used to probe the physiological roles of these proteases. A high degree of homology between family members and overlap in substrate specificity have made elucidating individual protease function, expression and activity difficult.Results: Using peptide vinyl

  3. 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

  4. Intracellular drug distribution-based targeting: Exploiting lysosomes to enhance the selectivity of drugs towards cancer cells

    E-print Network

    Ndolo, Rosemary A.

    2012-08-31

    mechanism of action [14]. For instance, it was due to the toxic effects of the chemical weapon sulfur mustard, originally synthesized in 1854 and used in World War I that anticancer drugs were discovered [15]. In World War II soldiers accidentally exposed... to sulfur mustard at Bari Harbor, Italy suffered from severe irritation of the respiratory tract and eye, and it was soon recognized that the toxic effects of sulfur mustard targeted the rapidly dividing cells of the gastrointestinal tract and blood...

  5. Lysosomal pH rise during heat shock monitored by a lysosome-targeting near-infrared ratiometric fluorescent probe.

    PubMed

    Wan, Qiongqiong; Chen, Suming; Shi, Wen; Li, Lihong; Ma, Huimin

    2014-10-01

    Heat stroke is a life-threatening condition, featuring a high body temperature and malfunction of many organ systems. The relationship between heat shock and lysosomes is poorly understood, mainly because of the lack of a suitable research approach. Herein, by incorporating morpholine into a stable hemicyanine skeleton, we develop a new lysosome-targeting near-infrared ratiometric pH?probe. In combination with fluorescence imaging, we show for the first time that the lysosomal pH?value increases but never decreases during heat shock, which might result from lysosomal membrane permeabilization. We also demonstrate that this lysosomal pH?rise is irreversible in living cells. Moreover, the probe is easy to synthesize, and shows superior overall analytical performance as compared to the existing commercial ones. This enhanced performance may enable it to be widely used in more lysosomal models of living cells and in further revealing the mechanisms underlying heat-related pathology. PMID:25154475

  6. Design of a simultaneous target and location-activatable fluorescent probe for visualizing hydrogen sulfide in lysosomes.

    PubMed

    Yang, Sheng; Qi, Yue; Liu, Changhui; Wang, Yijun; Zhao, Yirong; Wang, Lili; Li, Jishan; Tan, Weihong; Yang, Ronghua

    2014-08-01

    Molecular tools capable of providing information on a target analyte in an organelle of interest are especially appreciated. Traditionally, organelle-targetable probes are designed by incorporating an organelle-specific guiding unit to target the probe molecules into the organelle. The imperfect targeting function of the guiding unit and nonspecific distribution of the analyte in cytosol and each organelle would lead to low spatiotemporal resolution and limited sensitivity. To solve this problem, we report herein a new approach for detection of a target analyte in a specific organelle by engineering a target and location dual-controlled molecular switch. For this proof-of-concept study, fluorescent detection of H2S in lysosomes was performed with a simultaneous H2S and proton-activatable probe based on the acidic environment of lysosomes. The new synthesized fluorescent sensor, "SulpHensor", which contains a spirolactam moiety to bind hydrogen protons and an azide group to react with H2S, displays highly sensitive and selective fluorescence response to H2S under lysosomal pH environment but is out of operation in neutral cytosol and other organelles. Fluorescence imaging shows that SulpHensor is membrane-permeable and suitable for visualization of both the exogenous and endogenous H2S in lysosomes of living cells. The good performance of our proposed approach for H2S sensing demonstrates that this strategy might open up new opportunities for the development of efficient subcellular molecular tools for bioanalytical and biomedical applications. PMID:24975419

  7. 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

  8. 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

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

    PubMed

    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

  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 Ewing’s 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 µM, 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. Lysosome sorting of ?-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 ?-glucocerebrosidase (?-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 ?-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 ?-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 ?-GCase. The binding sites for ?-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-?-GCase lysosomal targeting. PMID:25027712

  12. Lysosome sorting of ?-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 ?-glucocerebrosidase (?-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 ?-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 ?-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 ?-GCase. The binding sites for ?-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–?-GCase lysosomal targeting. PMID:25027712

  13. In vitro and in vivo evaluation of a non-carbohydrate targeting platform for lysosomal proteins.

    PubMed

    Stefano, James E; Hou, Lihui; Honey, Denise; Kyazike, Josephine; Park, Anna; Zhou, Qun; Pan, Clark Q; Edmunds, Tim

    2009-04-17

    Lysosomal storage diseases arise from a genetic loss-of-function defect in enzymes mediating key catabolic steps resulting in accumulation of substrate within the lysosome. Treatment of several of these disorders has been achieved by enzyme replacement therapy (ERT), in which a recombinant version of the defective enzyme is expressed in vitro and administered by infusion. However, in many cases the biodistribution of the administered protein does not match that of the accumulated substrate due to the glycosylation-mediated clearance of the enzymes from circulation, resulting in poor or absent substrate clearance from some tissues. To overcome this limitation, we have evaluated several peptide-based targeting motifs to redirect recombinant human alpha-galactosidase (rhalphaGal) to specific receptors. A reversible thiol-based PEGylation chemistry was developed to achieve multivalent peptide display with lysosomal release. In vitro, cell uptake was peptide dependent and independent of the normal mannose-6-phosphate receptor mediated pathway. Surprisingly, despite increased plasma half-life and decreased liver uptake, none of the peptide conjugates showed significantly altered biodistribution in alphaGal-knockout mice. This suggests that these peptide-based targeting motifs are unlikely to provide substantial therapeutic benefit likely due to the complexity of factors affecting PK and biodistribution. PMID:19146893

  14. Dysfunction of two lysosome degradation pathways of ?-synuclein in Parkinson's disease: potential therapeutic targets?

    PubMed

    Jiang, Tian-Fang; Chen, Sheng-Di

    2012-10-01

    Parkinson's disease (PD) is pathologically characterized by the presence of ?-synuclein (?-syn)-positive intracytoplasmic inclusions named Lewy bodies in the dopaminergic neurons of the substantia nigra. A series of morbid consequences are caused by pathologically high amounts or mutant forms of ?-syn, such as defects of membrane trafficking and lipid metabolism. In this review, we consider evidence that both point mutation and overexpression of ?-syn result in aberrant degradation in neurons and microglia, and this is associated with the autophagy-lysosome pathway and endosome-lysosome system, leading directly to pathological intracellular aggregation, abnormal externalization and re-internalization cycling (and, in turn, internalization and re-externalization), and exocytosis. Based on these pathological changes, an increasing number of researchers have focused on these new therapeutic targets, aiming at alleviating the pathological accumulation of ?-syn and re-establishing normal degradation. PMID:22961477

  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. 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)1–6) 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 US12–US21; 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

  17. Role of Adaptor Complex AP-3 in Targeting Wild-Type and Mutated CD63 to Lysosomes

    PubMed Central

    Rous, Brian A.; Reaves, Barbara J.; Ihrke, Gudrun; Briggs, John A.G.; Gray, Sally R.; Stephens, David J.; Banting, George; Luzio, J. Paul

    2002-01-01

    CD63 is a lysosomal membrane protein that belongs to the tetraspanin family. Its carboxyterminal cytoplasmic tail sequence contains the lysosomal targeting motif GYEVM. Strong, tyrosine-dependent interaction of the wild-type carboxyterminal tail of CD63 with the AP-3 adaptor subunit ?3 was observed using a yeast two-hybrid system. The strength of interaction of mutated tail sequences with ?3 correlated with the degree of lysosomal localization of similarly mutated human CD63 molecules in stably transfected normal rat kidney cells. Mutated CD63 containing the cytosolic tail sequence GYEVI, which interacted strongly with ?3 but not at all with ?2 in the yeast two-hybrid system, localized to lysosomes in transfected normal rat kidney and NIH-3T3 cells. In contrast, it localized to the cell surface in transfected cells of pearl and mocha mice, which have genetic defects in genes encoding subunits of AP-3, but to lysosomes in functionally rescued mocha cells expressing the ? subunit of AP-3. Thus, AP-3 is absolutely required for the delivery of this mutated CD63 to lysosomes. Using this AP-3–dependent mutant of CD63, we have shown that AP-3 functions in membrane traffic from the trans-Golgi network to lysosomes via an intracellular route that appears to bypass early endosomes. PMID:11907283

  18. 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.

  19. The role of lysosomes in the selective concentration of mineral elements. A microanalytical study.

    PubMed

    Berry, J P

    1996-05-01

    The role of the lysosome during the intracellular concentration of diverse mineral elements has been evidenced by the electron probe X-ray microanalysis (EPMA). This highly sensitive technique allows an in situ chemical analysis of any chemical element with an atomic number greater than 11, present in ultra-thin tissue sections. Therefore, it has been demonstrated by using this EPMA that 21 out of the 92 elements of the periodic table, once injected in a soluble form, were selectively concentrated within lysosomes of several types of mammalian cells. Amongst these 21 elements, 15 are concentrated and precipitated in an insoluble from in association with phosphorus whereas the other 6 are precipitated in association with sulphur. Amongst the 15 elements which precipitate with phosphorus in lysosomes, there are: 3 group IIIB elements of the periodic system, (aluminium, gallium and indium); the rare-earth elements (cerium, gadolinium, lanthanum, thulium and samarium); 2 group IVA elements (hafnium and zirconium), two actinides (uranium and thorium) and elements such as chromium and niobium. The 6 elements which precipitate with sulphur comprise the 3 group VIII elements of the classification (nickel, palladium, platinum) and the 3 group IB elements (copper, silver and gold). The mechanisms responsible for this selective concentration involve enzymatic processes and predominantly acid phosphatases for elements precipitating as phosphates and arylsulfatases for elements precipitating with sulphur. PMID:8793193

  20. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25684681

  2. Rab7 silencing prevents ?-opioid receptor lysosomal targeting and rescues opioid responsiveness to strengthen diabetic neuropathic pain therapy.

    PubMed

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

    2013-04-01

    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

  3. SETI target selection.

    PubMed

    Latham, D W; Soderblom, D R

    1995-01-01

    The NASA High Resolution Microwave Survey consists of two complementary elements: a Sky Survey of the entire sky to a moderate level of sensitivity; and a Targeted Search of nearby stars, one at a time, to a much deeper level of sensitivity. In this paper we propose strategies for target selection. We have two goals: to improve the chances of successful detection of signals from technical civilizations that inhabit planets around solar-type stars, and to minimize the chances of missing signals from unexpected sites. For the main Targeted Search survey of approximately 1000 nearby solar-type stars, we argue that the selection criteria should be heavily biased by what we know about the origin and evolution of life here on Earth. We propose that observations of stars with stellar companions orbiting near the habitable zone should be de-emphasized, because such companions would prevent the formation of habitable planets. We also propose that observations of stars younger than about three billion years should be de-emphasized in favor of older stars, because our own technical civilization took longer than three billion years to evolve here on Earth. To provide the information needed for the preparation of specific target lists, we have undertaken an inventory of a large sample of solar-type stars out to a distance of 60 pc, with the goal of characterizing the relevant astrophysical properties of these stars, especially their ages and companionship. To complement the main survey, we propose that a modest sample of the nearest stars should be observed without any selection biases whatsoever. Finally, we argue that efforts to identify stars with planetary systems should be expanded. If found, such systems should receive intensive scrutiny. PMID:11540737

  4. Lysosomal storage disorders: The cellular impact of lysosomal dysfunction

    PubMed Central

    2012-01-01

    Lysosomal storage diseases (LSDs) are a family of disorders that result from inherited gene mutations that perturb lysosomal homeostasis. LSDs mainly stem from deficiencies in lysosomal enzymes, but also in some non-enzymatic lysosomal proteins, which lead to abnormal storage of macromolecular substrates. Valuable insights into lysosome functions have emerged from research into these diseases. In addition to primary lysosomal dysfunction, cellular pathways associated with other membrane-bound organelles are perturbed in these disorders. Through selective examples, we illustrate why the term “cellular storage disorders” may be a more appropriate description of these diseases and discuss therapies that can alleviate storage and restore normal cellular function. PMID:23185029

  5. Lysosomal physiology.

    PubMed

    Xu, Haoxing; Ren, Dejian

    2015-02-10

    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

  6. BCA2/Rabring7 Targets HIV-1 Gag for Lysosomal Degradation in a Tetherin-Independent Manner

    PubMed Central

    Nityanandam, Ramya; Serra-Moreno, Ruth

    2014-01-01

    BCA2 (Rabring7, RNF115 or ZNF364) is a RING-finger E3 ubiquitin ligase that was identified as a co-factor in the restriction imposed by tetherin/BST2 on HIV-1. Contrary to the current model, in which BCA2 lacks antiviral activity in the absence of tetherin, we found that BCA2 possesses tetherin-independent antiviral activity. Here we show that the N-terminus of BCA2 physically interacts with the Matrix region of HIV-1 and other retroviral Gag proteins and promotes their ubiquitination, redistribution to endo-lysosomal compartments and, ultimately, lysosomal degradation. The targeted depletion of BCA2 in tetherin-expressing and tetherin-deficient cells results in a significant increase in virus release and replication, indicating that endogenous BCA2 possesses antiviral activity. Therefore, these results indicate that BCA2 functions as an antiviral factor that targets HIV-1 Gag for degradation, impairing virus assembly and release. PMID:24852021

  7. 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 next–generation-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

  8. Enhanced delivery of ?-glucosidase for Pompe disease by ICAM-1-targeted nanocarriers: comparative performance of a strategy for three distinct lysosomal storage disorders

    PubMed Central

    Hsu, Janet; Northrup, Laura; Bhowmick, Tridib; Muro, Silvia

    2011-01-01

    Enzyme replacement therapies for lysosomal storage disorders are often hindered by suboptimal biodistribution of recombinant enzymes after systemic injection. This is the case for Pompe disease caused by acid ?-glucosidase (GAA) deficiency, leading to excess glycogen storage through the body, mainly the liver and striated muscle. Targeting intercellular adhesion molecule-1 (ICAM-1), a protein involved in inflammation and overexpressed on most cells under pathological conditions, provides broad biodistribution and lysosomal transport of therapeutic cargoes. To improve its delivery, we coupled GAA to polymer nanocarriers (~180-nm) coated with anti-ICAM. Fluorescence microscopy showed specific targeting of anti-ICAM/GAA NCs to cells, with efficient internalization and lysosomal transport, enhancing glycogen degradation over non-targeted GAA. Radioisotope tracing in mice demonstrated enhanced GAA accumulation in all organs, including Pompe targets. Along with improved delivery of Niemann-Pick and Fabry enzymes, previously described, these results indicate that ICAM-1 targeting holds promise as a broad platform for lysosomal enzyme delivery. PMID:21906578

  9. Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein.

    PubMed

    Kiss, Katalin; Kucsma, Nora; Brozik, Anna; Tusnady, Gabor E; Bergam, Ptissam; van Niel, Guillaume; Szakacs, Gergely

    2015-04-01

    ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core-ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core-ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6. PMID:25627919

  10. Comet and Target Ghost: Techniques for Selecting Moving Targets

    E-print Network

    Comet and Target Ghost: Techniques for Selecting Moving Targets Khalad Hasan1 , Tovi Grossman2 that assist in selecting moving targets. We present Comet, a technique that enhances tar- gets based of the target, while leaving the motion uninterrupted. We found a speed benefit for the Comet in a 1D selection

  11. Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

    PubMed Central

    Bartee, Eric; Eyster, Craig A.; Viswanathan, Kasinath; Mansouri, Mandana; Donaldson, Julie G.; Früh, Klaus

    2010-01-01

    Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins. PMID:21151997

  12. Ethical Implications of Target Market Selection

    Microsoft Academic Search

    Terri L. Rittenburg; Madhavan Parthasarathy

    1997-01-01

    Marketers have been criticized recently for the selection of target markets, especially for targeting disadvantaged segments of a society with harmful products. Very little has been done, however, to provide guidelines for marketers developing target market strategies. This article examines the ethical dimensions of target market selection. The proposed model for analyzing target markets allows for differences in both the

  13. Characterization of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Trafficking Reveals a Novel Lysosomal Targeting Mechanism via Amyloid Precursor-like Protein 2 (APLP2)

    PubMed Central

    DeVay, Rachel M.; Shelton, David L.; Liang, Hong

    2013-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor protein levels by diverting it to lysosomes. Monoclonal antibody therapeutics aimed to neutralize PCSK9 have been shown to successfully lower serum LDL levels; however, we previously found that such therapeutic antibodies are subject to PCSK9-mediated clearance. In this study, we discovered that PCSK9 interacts via its C-terminal domain directly and in a pH-dependent manner with amyloid precursor protein as well as its closely related family member, amyloid precursor protein-like protein 2. Furthermore, we determined that amyloid precursor protein-like protein-2, but not amyloid precursor protein, is involved in mediating postendocytic delivery of PCSK9 to lysosomes and is therefore important for PCSK9 function. Based on our data, we propose a model for a lysosomal transport complex by which a soluble protein can target another protein for degradation from the luminal side of the membrane by bridging it to a lysosomally targeted transmembrane protein. PMID:23430252

  14. Fusion to the Lysosome Targeting Signal of the Invariant Chain Alters the Processing and Enhances the Immunogenicity of HIV-1 Reverse Transcriptase.

    PubMed

    Starodubova, E S; Isaguliants, M G; Kuzmenko, Y V; Latanova, A A; Krotova, O A; Karpov, V L

    2014-01-01

    Intracellular processing of the antigen encoded by a DNA vaccine is one of the key steps in generating an immune response. Immunization with DNA constructs targeted to the endosomal-lysosomal compartments and to the MHC class II pathway can elicit a strong immune response. Herein, the weakly immunogenic reverse transcriptase of HIV-1 was fused to the minimal lysosomal targeting motif of the human MHC class II invariant chain. The motif fused to the N-terminus shifted the enzyme intracellular localization and accelerated its degradation. Degradation of the chimeric protein occurred predominantly in the lysosomal compartment. BALB/c mice immunized with the plasmid encoding the chimeric protein demonstrated an enhanced immune response, in the form of an increased antigen-specific production of Th1 cytokines, INF-? and IL-2, by mouse splenocytes. Moreover, the majority of the splenocytes secreted both cytokines; i.e., were polyfunctional. These findings suggest that retargeting of the antigen to the lysosomes enhances the immune response to DNA vaccine candidates with low intrinsic immunogenicity. PMID:24772328

  15. Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

    PubMed Central

    Balut, Corina M.; Loch, Christian M.; Devor, Daniel C.

    2011-01-01

    We recently demonstrated that plasma membrane KCa3.1 is rapidly endocytosed and targeted for lysosomal degradation via a Rab7- and ESCRT-dependent pathway. Herein, we assess the role of ubiquitylation in this process. Using a biotin ligase acceptor peptide (BLAP)-tagged KCa3.1, in combination with tandem ubiquitin binding entities (TUBEs), we demonstrate that KCa3.1 is polyubiquitylated following endocytosis. Hypertonic sucrose inhibited KCa3.1 endocytosis and resulted in a significant decrease in channel ubiquitylation. Inhibition of the ubiquitin-activating enzyme (E1) with UBEI-41 resulted in reduced KCa3.1 ubiquitylation and internalization. The general deubiquitylase (DUB) inhibitor, PR-619 attenuated KCa3.1 degradation, indicative of deubiquitylation being required for lysosomal delivery. Using the DUB Chip, a protein microarray containing 35 DUBs, we demonstrate a time-dependent association between KCa3.1 and USP8 following endocytosis, which was confirmed by coimmunoprecipitation. Further, overexpression of wild-type USP8 accelerates channel deubiquitylation, while either a catalytically inactive mutant USP8 or siRNA-mediated knockdown of USP8 enhanced accumulation of ubiquitylated KCa3.1, thereby inhibiting channel degradation. In summary, by combining BLAP-tagged KCa3.1 with TUBEs and DUB Chip methodologies, we demonstrate that polyubiquitylation mediates the targeting of membrane KCa3.1 to the lysosomes and also that USP8 regulates the rate of KCa3.1 degradation by deubiquitylating KCa3.1 prior to lysosomal delivery.—Balut, C. M., Loch, C. M., Devor, D. C. Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1. PMID:21828287

  16. Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase.

    PubMed

    Petersen, Nikolaj H T; Olsen, Ole D; Groth-Pedersen, Line; Ellegaard, Anne-Marie; Bilgin, Mesut; Redmer, Susanne; Ostenfeld, Marie S; Ulanet, Danielle; Dovmark, Tobias H; Lønborg, Andreas; Vindeløv, Signe D; Hanahan, Douglas; Arenz, Christoph; Ejsing, Christer S; Kirkegaard, Thomas; Rohde, Mikkel; Nylandsted, Jesper; Jäättelä, Marja

    2013-09-01

    Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert multidrug resistance. Their cancer selectivity is associated with transformation-associated reduction in ASM expression and subsequent failure to maintain sphingomyelin hydrolysis during drug exposure. Taken together, these data identify ASM as an attractive target for cancer therapy. PMID:24029234

  17. The Mechanical Activation of mTOR Signaling: An Emerging Role for Late Endosome/Lysosomal Targeting

    PubMed Central

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

    2013-01-01

    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

  18. BODIPY-based fluorescent thermometer as a lysosome-targetable probe: how the oligo(ethylene glycols) compete photoinduced electron transfer.

    PubMed

    Wang, Hua; Wu, Yongquan; Shi, Yanlin; Tao, Pan; Fan, Xing; Su, Xinyan; Kuang, Gui-Chao

    2015-02-16

    A novel BODIPY-based fluorescent thermometer, which shows a lysosome-targeting property, was successfully prepared. Due to the electron-donating ability of the oligo(ethylene glycols), the photoinduced electron-transfer pathway from morpholine to BODIPY dye is blocked. The fluorescence of the thermometer quenched by intramolecular rotation at room temperature was progressively enhanced during heating due to the increased microviscosity around the fluorophore. PMID:25557269

  19. 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 ?-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

  20. Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance

    PubMed Central

    Zhitomirsky, Benny; Assaraf, Yehuda G.

    2015-01-01

    Multidrug resistance (MDR) is a primary hindrance to curative cancer chemotherapy. In this respect, lysosomes were suggested to play a role in intrinsic MDR by sequestering protonated hydrophobic weak base chemotherapeutics away from their intracellular target sites. Here we show that intrinsic resistance to sunitinib, a hydrophobic weak base tyrosine kinase inhibitor known to accumulate in lysosomes, tightly correlates with the number of lysosomes accumulating high levels of sunitinib in multiple human carcinoma cells. Furthermore, exposure of cancer cells to hydrophobic weak base drugs leads to a marked increase in the number of lysosomes per cell. Non-cytotoxic, nanomolar concentrations, of the hydrophobic weak base chemotherapeutics doxorubicin and mitoxantrone triggered rapid lysosomal biogenesis that was associated with nuclear translocation of TFEB, the dominant transcription factor regulating lysosomal biogenesis. This resulted in increased lysosomal gene expression and lysosomal enzyme activity. Thus, treatment of cancer cells with hydrophobic weak base chemotherapeutics and their consequent sequestration in lysosomes triggers lysosomal biogenesis, thereby further enhancing lysosomal drug entrapment and MDR. The current study provides the first evidence that drug-induced TFEB-associated lysosomal biogenesis is an emerging determinant of MDR and suggests that circumvention of lysosomal drug sequestration is a novel strategy to overcome this chemoresistance. PMID:25544758

  1. Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1.

    PubMed

    Balut, Corina M; Loch, Christian M; Devor, Daniel C

    2011-11-01

    We recently demonstrated that plasma membrane KCa3.1 is rapidly endocytosed and targeted for lysosomal degradation via a Rab7- and ESCRT-dependent pathway. Herein, we assess the role of ubiquitylation in this process. Using a biotin ligase acceptor peptide (BLAP)-tagged KCa3.1, in combination with tandem ubiquitin binding entities (TUBEs), we demonstrate that KCa3.1 is polyubiquitylated following endocytosis. Hypertonic sucrose inhibited KCa3.1 endocytosis and resulted in a significant decrease in channel ubiquitylation. Inhibition of the ubiquitin-activating enzyme (E1) with UBEI-41 resulted in reduced KCa3.1 ubiquitylation and internalization. The general deubiquitylase (DUB) inhibitor, PR-619 attenuated KCa3.1 degradation, indicative of deubiquitylation being required for lysosomal delivery. Using the DUB Chip, a protein microarray containing 35 DUBs, we demonstrate a time-dependent association between KCa3.1 and USP8 following endocytosis, which was confirmed by coimmunoprecipitation. Further, overexpression of wild-type USP8 accelerates channel deubiquitylation, while either a catalytically inactive mutant USP8 or siRNA-mediated knockdown of USP8 enhanced accumulation of ubiquitylated KCa3.1, thereby inhibiting channel degradation. In summary, by combining BLAP-tagged KCa3.1 with TUBEs and DUB Chip methodologies, we demonstrate that polyubiquitylation mediates the targeting of membrane KCa3.1 to the lysosomes and also that USP8 regulates the rate of KCa3.1 degradation by deubiquitylating KCa3.1 prior to lysosomal delivery. PMID:21828287

  2. Inactivation of the β-subunit of lysosomal β-hexosaminidase by targeted disruption of the HEXB gene in mice

    Microsoft Academic Search

    D. Phaneuf; J. Trasler; R. A. Gravel

    1994-01-01

    β-Hexosaminidase is a lysosomal enzyme that catalyzes the hydrolysis of ganglioside G{sub M2} and other substances. Two major catalytically active isozymes are expressed in human tissues: Hex A (aβ) and Hex B (ββ). Sandhoff disease is a rare autosomal recessive disorder caused by mutations in the HEXB gene, inactivating both isoenzymes. We have characterized the structure of the mouse HEXB

  3. Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice.

    PubMed

    Kollmann, K; Damme, M; Markmann, S; Morelle, W; Schweizer, M; Hermans-Borgmeyer, I; Röchert, A K; Pohl, S; Lübke, T; Michalski, J-C; Käkelä, R; Walkley, S U; Braulke, T

    2012-09-01

    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 Niemann-Pick 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

  4. Agilent Technologies SureSelect Target

    E-print Network

    Kopp, Artyom

    Agilent Technologies SureSelect Target Enrichment System Illumina Paired-End Sequencing Platform Library Prep Protocol Version 1.0, September 2009 SureSelect platform manufactured with Agilent Sure Enrichment System Protocol Notices © Agilent Technologies, Inc. 2009 No part of this manual may be reproduced

  5. A parasitic helminth-derived peptide that targets the macrophage lysosome is a novel therapeutic option for autoimmune disease.

    PubMed

    Alvarado, Raquel; O'Brien, Bronwyn; Tanaka, Akane; Dalton, John P; Donnelly, Sheila

    2015-02-01

    Parasitic worms (helminths) reside in their mammalian hosts for many years. This is attributable, in part, to their ability to skew the host's immune system away from pro-inflammatory responses and towards anti-inflammatory or regulatory responses. This immune modulatory ability ensures helminth longevity within the host, while simultaneously minimises tissue destruction for the host. The molecules that the parasite releases clearly exert potent immune-modulatory actions, which could be exploited clinically, for example in the prophylactic and therapeutic treatment of pro-inflammatory and autoimmune diseases. We have identified a novel family of immune-modulatory proteins, termed helminth defence molecules (HDMs), which are secreted by several medically important helminth parasites. These HDMs share biochemical and structural characteristics with mammalian cathelicidin-like host defence peptides (HDPs), which are significant components of the innate immune system. Like their mammalian counterparts, parasite HDMs block the activation of macrophages via toll like receptor (TLR) 4 signalling, however HDMs are significantly less cytotoxic than HDPs. HDMs can traverse the cell membrane of macrophages and enter the endolysosomal system where they reduce the acidification of lysosomal compartments by inhibiting vacuolar (v)-ATPase activity. In doing this, HDMs can modulate critical cellular functions, such as cytokine secretion and antigen processing/presentation. Here, we review the role of macrophages, specifically their lysosomal mediated activities, in the initiation and perpetuation of pro-inflammatory immune responses. We also discuss the potential of helminth defence molecules (HDMs) as therapeutics to counteract the pro-inflammatory responses underlying autoimmune disease. Given the current lack of effective, non-cytotoxic treatment options to limit the progression of autoimmune pathologies, HDMs open novel treatment avenues. PMID:25466586

  6. 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

  7. 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.

  8. Optimal adaptive waveform selection for target tracking

    Microsoft Academic Search

    B. L. Scala; M. Rezaeian; B. Moran

    2005-01-01

    Modern phased array radars, with flexible waveform generation and beam steering capability, are able to adaptively modify their performance to suit a variety of environments. This power has not yet been fully exploited, in part because of the lack of suitable scheduling algorithms. This paper describes an optimal adaptive waveform selection algorithm for target tracking. An adaptive scheduling algorithm that

  9. 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

  10. Target Goals--Which Ones Are Schools Selecting?

    ERIC Educational Resources Information Center

    Babich, George; Hunter, David

    1996-01-01

    Examines the target area goals selected by 69 elementary, middle, and secondary schools that were candidates for outcomes accreditation in Arizona in 1994-95. Describes the target area goals and discusses the most frequently selected goals, the distribution of goals among target areas, and distributions within selected target areas. (MAB)

  11. 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.

  12. Selecting Potential Targetable Biomarkers for Imaging Purposes in Colorectal Cancer Using TArget Selection Criteria (TASC): A Novel Target Identification Tool

    PubMed Central

    van Oosten, Marleen; Crane, Lucia MA; Bart, Joost; van Leeuwen, Fijs W; van Dam, Gooitzen M

    2011-01-01

    Peritoneal carcinomatosis (PC) of colorectal origin is associated with a poor prognosis. However, cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy is available for a selected group of PC patients, which significantly increases overall survival rates up to 30%. As a consequence, there is substantial room for improvement. Tumor targeting is expected to improve the treatment efficacy of colorectal cancer (CRC) further through 1) more sensitive preoperative tumor detection, thus reducing overtreatment; 2) better intraoperative detection and surgical elimination of residual disease using tumor-specific intraoperative imaging; and 3) tumor-specific targeted therapeutics. This review focuses, in particular, on the development of tumor-targeted imaging agents. A large number of biomarkers are known to be upregulated in CRC. However, to date, no validated criteria have been described for the selection of the most promising biomarkers for tumor targeting. Such a scoring system might improve the selection of the correct biomarker for imaging purposes. In this review, we present the TArget Selection Criteria (TASC) scoring system for selection of potential biomarkers for tumor-targeted imaging. By applying TASC to biomarkers for CRC, we identified seven biomarkers (carcinoembryonic antigen, CXC chemokine receptor 4, epidermal growth factor receptor, epithelial cell adhesion molecule, matrix metalloproteinases, mucin 1, and vascular endothelial growth factor A) that seem most suitable for tumor-targeted imaging applications in colorectal cancer. Further cross-validation studies in CRC and other tumor types are necessary to establish its definitive value. PMID:21461170

  13. mTOR and lysosome regulation

    PubMed Central

    2014-01-01

    Lysosomes are key cellular organelles that play a crucial role in catabolism by degrading extracellular and intracellular material. It is, therefore, very intriguing that mTORC1 (mechanistic target of rapamycin complex 1), a major promoter of anabolic processes, localizes in its active form to the surface of lysosomes. In recent years, many exciting observations have revealed a tightly regulated crosstalk between mTORC1 activity and lysosomal function. These findings highlight the complex regulatory network that modulates energy metabolism in cells. PMID:25184042

  14. A Color Hierarchy for Automatic Target Selection

    PubMed Central

    Tchernikov, Illia; Fallah, Mazyar

    2010-01-01

    Visual processing of color starts at the cones in the retina and continues through ventral stream visual areas, called the parvocellular pathway. Motion processing also starts in the retina but continues through dorsal stream visual areas, called the magnocellular system. Color and motion processing are functionally and anatomically discrete. Previously, motion processing areas MT and MST have been shown to have no color selectivity to a moving stimulus; the neurons were colorblind whenever color was presented along with motion. This occurs when the stimuli are luminance-defined versus the background and is considered achromatic motion processing. Is motion processing independent of color processing? We find that motion processing is intrinsically modulated by color. Color modulated smooth pursuit eye movements produced upon saccading to an aperture containing a surface of coherently moving dots upon a black background. Furthermore, when two surfaces that differed in color were present, one surface was automatically selected based upon a color hierarchy. The strength of that selection depended upon the distance between the two colors in color space. A quantifiable color hierarchy for automatic target selection has wide-ranging implications from sports to advertising to human-computer interfaces. PMID:20195361

  15. Nonresonant and Resonant Frequency-Selectable Induction-Heating Targets

    E-print Network

    Rodriguez, John I.

    This paper examines a scheme for developing frequency-selectable induction-heating targets for stimulating temperature-sensitive polymer gels. The phrase “frequency selectable” implies that each target has a frequency at ...

  16. Internalizing MHC class II–peptide complexes are ubiquitinated in early endosomes and targeted for lysosomal degradation

    PubMed Central

    Furuta, Kazuyuki; Walseng, Even; Roche, Paul A.

    2013-01-01

    As sentinels of the immune system, dendritic cells (DCs) continuously generate and turnover antigenic peptide–MHC class II complexes (pMHC-II). pMHC-II generation is a complex process that involves many well-characterized MHC-II biosynthetic intermediates; however, the mechanisms leading to MHC-II turnover/degradation are poorly understood. We now show that pMHC-II complexes undergoing clathrin-independent endocytosis from the DC surface are efficiently ubiquitinated by the E3 ubiquitin ligase March-I in early endosomes, whereas biosynthetically immature MHC-II–Invariant chain (Ii) complexes are not. The inability of MHC-II–Ii to serve as a March-I substrate is a consequence of Ii sorting motifs that divert the MHC-II–Ii complex away from March-I+ early endosomes. When these sorting motifs are mutated, or when clathrin-mediated endocytosis is inhibited, MHC-II–Ii complexes internalize by using a clathrin-independent endocytosis pathway and are now ubiquitinated as efficiently as pMHC-II complexes. These data show that the selective ubiquitination of internalizing surface pMHC-II in March-I+ early endosomes promotes degradation of “old” pMHC-II and spares forms of MHC-II that have not yet loaded antigenic peptides or have not yet reached the DC surface. PMID:24277838

  17. 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

  18. Selective targeting of microglia by quantum dots

    PubMed Central

    2012-01-01

    Background Microglia, the resident immune cells of the brain, have been implicated in brain injury and various neurological disorders. However, their precise roles in different pathophysiological situations remain enigmatic and may range from detrimental to protective. Targeting the delivery of biologically active compounds to microglia could help elucidate these roles and facilitate the therapeutic modulation of microglial functions in neurological diseases. Methods Here we employ primary cell cultures and stereotaxic injections into mouse brain to investigate the cell type specific localization of semiconductor quantum dots (QDs) in vitro and in vivo. Two potential receptors for QDs are identified using pharmacological inhibitors and neutralizing antibodies. Results In mixed primary cortical cultures, QDs were selectively taken up by microglia; this uptake was decreased by inhibitors of clathrin-dependent endocytosis, implicating the endosomal pathway as the major route of entry for QDs into microglia. Furthermore, inhibiting mannose receptors and macrophage scavenger receptors blocked the uptake of QDs by microglia, indicating that QD uptake occurs through microglia-specific receptor endocytosis. When injected into the brain, QDs were taken up primarily by microglia and with high efficiency. In primary cortical cultures, QDs conjugated to the toxin saporin depleted microglia in mixed primary cortical cultures, protecting neurons in these cultures against amyloid beta-induced neurotoxicity. Conclusions These findings demonstrate that QDs can be used to specifically label and modulate microglia in primary cortical cultures and in brain and may allow for the selective delivery of therapeutic agents to these cells. PMID:22272874

  19. Lysosomal storage disorders

    Microsoft Academic Search

    Ashok Vellodi

    2005-01-01

    Summary Although the first description of a lysosomal storage disorder was that of Tay-Sachs disease in 1881, the lysosome was not discovered until 1955, by Christian De Duve. The first demonstration by Hers in 1963 of a link between an enzyme deficiency and a storage disorder (Pompe's disease) paved the way for a series of seminal discoveries about the intracellular

  20. Lysosomal storage diseases: from pathophysiology to therapy.

    PubMed

    Parenti, Giancarlo; Andria, Generoso; Ballabio, Andrea

    2015-01-01

    Lysosomal storage diseases are a group of rare, inborn, metabolic errors characterized by deficiencies in normal lysosomal function and by intralysosomal accumulation of undegraded substrates. The past 25 years have been characterized by remarkable progress in the treatment of these diseases and by the development of multiple therapeutic approaches. These approaches include strategies aimed at increasing the residual activity of a missing enzyme (enzyme replacement therapy, hematopoietic stem cell transplantation, pharmacological chaperone therapy and gene therapy) and approaches based on reducing the flux of substrates to lysosomes. As knowledge has improved about the pathophysiology of lysosomal storage diseases, novel targets for therapy have been identified, and innovative treatment approaches are being developed. PMID:25587658

  1. [Pseudodeficiency of lysosomal enzymes].

    PubMed

    Czartoryska, B

    1995-01-01

    Genetically determined enzyme deficiency causing failure of the lysosomal apparatus is called lysosomal disease. In normal cell the activity of lysosomal enzymes exceeds many times the requirements of the cell. In some individuals due to gene mutation the activity of an intracellular enzyme is only slightly higher than in patients with lysosomal disease but much lower than in the general population, although without evident metabolic and clinical consequences. This situation is called enzyme pseudodeficiency. As yet cases have been reported of the pseudodeficiency of beta-galactocerebrosidase, beta-glucoronidase, beta-glucosidase, beta-hexosoaminidase A and arylosulfatase A. The character of the mutation is called in the case of the two last enzymes and a laboratory method is available for differentiation of pseudodeficiency from the actual lysosomal disease. It is not known whether in pseudodeficiency of an enzyme clinical manifestations could appear in older age. PMID:7596481

  2. Clarifying lysosomal storage diseases

    PubMed Central

    Schultz, Mark L; Tecedor, Luis; Chang, Michael; Davidson, Beverly L.

    2011-01-01

    Lysosomal storage diseases (LSDs) are a class of metabolic disorders caused by mutations in proteins critical for lysosomal function. Such proteins include lysosomal enzymes, lysosomal integral membrane proteins, and proteins involved in the post-translational modification and trafficking of lysosomal proteins. There are many recognized forms of LSDs, and although individually rare, their combined prevalence is estimated to be 1 in 8000 births. Over two-thirds of LSDs involve central nervous system (CNS) dysfunction—progressive cognitive and motor decline—and these symptoms are often the most debilitating. Although the genetic basis for these disorders are clear and the biochemistry of the proteins well understood, the cellular mechanisms by which deficiencies in these proteins disrupts neuronal viability remain ambiguous. In this review, we provide an overview of the widespread cellular perturbations occurring in LSDs, how they may be linked, and interventions that may specifically or globally correct those defects. PMID:21723623

  3. Lysosomotropic agents: impact on lysosomal membrane permeabilization and cell death.

    PubMed

    Villamil Giraldo, Ana M; Appelqvist, Hanna; Ederth, Thomas; Öllinger, Karin

    2014-10-01

    Lysosomes are acidic organelles essential for degradation, signalling and cell homoeostasis. In addition, they play a key role in cell death. Permeabilization of the lysosomal membrane and release of hydrolytic enzymes to the cytosol accompanies apoptosis signalling in several systems. The regulatory mechanism of lysosomal stability is, however, poorly understood. Lipophilic or amphiphilic compounds with a basic moiety will become protonated and trapped within lysosomes, and such lysosomotropic behaviour is also found in many pharmacological drugs. The natural sphingolipid sphingosine exhibits lysosomotropic detergent ability and is an endogenous candidate for controlling lysosomal membrane permeabilization. The lysosomotropic properties of certain detergents might be of use in lysosome-targeting anticancer drugs and drug delivery system in the future. The present review summarizes the current knowledge on the targeting and permeabilizing properties of lysosomotropic detergents from a cellular and physicochemical perspective. PMID:25233432

  4. Target Tracking with Online Feature Selection in FLIR Imagery

    Microsoft Academic Search

    Vijay Venkataraman; Guoliang Fan; Xin Fan

    2007-01-01

    We present a particle lter -based target tracking algo- rithm for FLIR imagery. A dual foreground and background model is proposed for target representation which supports robust and accurate target tracking and size estimation. A novel online feature selection technique is introduced that is able to adaptively select the optimal feature to maximize the tracking condence . Moreover, a coupled

  5. Aging. Lysosomal signaling molecules regulate longevity in Caenorhabditis elegans.

    PubMed

    Folick, Andrew; Oakley, Holly D; Yu, Yong; Armstrong, Eric H; Kumari, Manju; Sanor, Lucas; Moore, David D; Ortlund, Eric A; Zechner, Rudolf; Wang, Meng C

    2015-01-01

    Lysosomes are crucial cellular organelles for human health that function in digestion and recycling of extracellular and intracellular macromolecules. We describe a signaling role for lysosomes that affects aging. In the worm Caenorhabditis elegans, the lysosomal acid lipase LIPL-4 triggered nuclear translocalization of a lysosomal lipid chaperone LBP-8, which promoted longevity by activating the nuclear hormone receptors NHR-49 and NHR-80. We used high-throughput metabolomic analysis to identify several lipids in which abundance was increased in worms constitutively overexpressing LIPL-4. Among them, oleoylethanolamide directly bound to LBP-8 and NHR-80 proteins, activated transcription of target genes of NHR-49 and NHR-80, and promoted longevity in C. elegans. These findings reveal a lysosome-to-nucleus signaling pathway that promotes longevity and suggest a function of lysosomes as signaling organelles in metazoans. PMID:25554789

  6. 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 [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)] [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)] [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    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.

  7. Evaluating the targets of selection during character displacement.

    PubMed

    Martin, Ryan A; Pfennig, David W

    2011-10-01

    Ecological character displacement occurs when competition imposes divergent selection on interacting species, causing divergence in traits associated with resource use. Generally, divergence is assumed to occur when selection acts on the same, continuously varying trait in both species. However, selection might target multiple traits, and even closely related heterospecifics involved in character displacement might differ in selective targets. We investigated the targets of selection in a species of spadefoot toad, Spea multiplicata, during experimentally imposed competition with a congener, S. bombifrons. When examining traits separately, we found significant selection acting on multiple resource-acquisition traits. Yet, controlling for the independent effects of these traits in a multiple regression revealed that direct selection on a single trait might have contributed toward indirect selection on other correlated traits. Moreover, although we found evidence for plasticity in most traits, competition with S. bombifrons imposed selection on morphology and not on plasticity. Additional experiments suggest that the selective targets during character displacement might differ between the two species involved in this one instance of character displacement. Identifying the targets of competitively mediated selection is crucial, because whether and how character displacement ultimately unfolds depends on the nature of these targets and correlations among them. PMID:21967434

  8. Isofagomine increases lysosomal delivery of exogenous glucocerebrosidase

    Microsoft Academic Search

    Jin-Song Shen; Nancy J. Edwards; Young Bin Hong; Gary J. Murray

    2008-01-01

    Intravenous enzyme replacement therapy (ERT) with purified glucocerebrosidase (GLA) leads to significant improvement of the clinical manifestations in patients with Type 1 Gaucher disease. However, the high doses required, slow response and inability to recover most of the infused enzyme in the target tissues may be attributed to losses occurring during transit en route to the lysosome. Preincubation of GLA

  9. UVA causes dual inactivation of cathepsin B and L underlying lysosomal dysfunction in human dermal fibroblasts.

    PubMed

    Lamore, Sarah D; Wondrak, Georg T

    2013-06-01

    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

  10. Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

    Microsoft Academic Search

    Katy Janvier; Juan S. Bonifacino

    2005-01-01

    The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However,

  11. 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

  12. Target selection bias transfers across different response actions.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2014-06-01

    Target selection is biased by recent experience. For example, a selected target feature may be stored in memory and bias selection on future trials, such that objects matching that feature are "primed" for selection. In the present study, we examined the role of action history in selection biases. Participants searched for a uniquely colored object. Pretrial cues indicated whether participants should respond with a keypress or a reach movement. If the representation of the feature that biases selection is critically bound with its associated action, we would expect priming effects to be restricted to cases where both the response mode and target color are repeated. However, we found that responses to the target were faster when the target color was repeated, even when the response switched from a reach to a keypress, or vice versa. Priming effects were even observed after "no-go" trials in which a response was withheld, and priming effects transferred across response modes when eye movement recordings ensured that participants did not saccade to the target. These results demonstrate that target features are represented in memory separately from their associated actions and can bias selection on subsequent trials even when a different mode of action output is required. PMID:24490945

  13. Computational approaches to selecting and optimising targets for structural biology

    PubMed Central

    Overton, Ian M.; Barton, Geoffrey J.

    2011-01-01

    Selection of protein targets for study is central to structural biology and may be influenced by numerous factors. A key aim is to maximise returns for effort invested by identifying proteins with the balance of biophysical properties that are conducive to success at all stages (e.g. solubility, crystallisation) in the route towards a high resolution structural model. Selected targets can be optimised through construct design (e.g. to minimise protein disorder), switching to a homologous protein, and selection of experimental methodology (e.g. choice of expression system) to prime for efficient progress through the structural proteomics pipeline. Here we discuss computational techniques in target selection and optimisation, with more detailed focus on tools developed within the Scottish Structural Proteomics Facility (SSPF); namely XANNpred, ParCrys, OB-Score (target selection) and TarO (target optimisation). TarO runs a large number of algorithms, searching for homologues and annotating the pool of possible alternative targets. This pool of putative homologues is presented in a ranked, tabulated format and results are also visualised as an automatically generated and annotated multiple sequence alignment. The target selection algorithms each predict the propensity of a selected protein target to progress through the experimental stages leading to diffracting crystals. This single predictor approach has advantages for target selection, when compared with an approach using two or more predictors that each predict for success at a single experimental stage. The tools described here helped SSPF achieve a high (21%) success rate in progressing cloned targets to diffraction-quality crystals. PMID:21906678

  14. Optimal adaptive waveform selection for target detection

    Microsoft Academic Search

    B. F. La Scala; W. Moran; R. J. Evans

    2003-01-01

    Modern phased array radars are able to adaptively modify their performance to the environment. To make full use of this capability, scheduling algorithms need to be designed. This paper poses the problem of adaptive waveform scheduling for detecting new targets in the context of finite horizon stochastic dynamic programming. The result is a scheduling algorithm that minimises the time taken

  15. Lysosome: regulator of lipid degradation pathways

    PubMed Central

    Settembre, Carmine; Ballabio, Andrea

    2014-01-01

    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

  16. General lysosomal hydrolysis can process prorenin accurately.

    PubMed

    Xa, Lucie K; Lacombe, Marie-Josée; 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. 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

  18. Lysosomal Lipid Storage Diseases

    PubMed Central

    Schulze, Heike; Sandhoff, Konrad

    2011-01-01

    Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a “traffic jam.” This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement. PMID:21502308

  19. Sexual selection targets cetacean pelvic bones.

    PubMed

    Dines, James P; Otárola-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

  20. High affinity ligands from in vitro selection: Complex?targets

    PubMed Central

    Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

    1998-01-01

    Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

  1. Target selection and current status of structural genomics for the

    E-print Network

    Babu, M. Madan

    33 Target selection and current status of structural genomics for the completed microbial genomes 3.2 Structural status of completed microbial genomes in the PDB................ 3.3 Metabolic pathways as targets for structural genomics.......................... 3.3.1 Glycolytic pathway

  2. Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology

    NSDL National Science Digital Library

    Emma J. Parkinson-Lawrence (South Australian Pathology Services)

    2010-04-01

    The discovery over five decades ago of the lysosome, as a degradative organelle and its dysfunction in lysosomal storage disorder patients, was both insightful and simple in concept. Here, we review some of the history and pathophysiology of lysosomal storage disorders to show how they have impacted on our knowledge of lysosomal biology. Although a significant amount of information has been accrued on the molecular genetics and biochemistry of lysosomal storage disorders, we still do not fully understand the mechanistic link between the storage material and disease pathogenesis. However, the accumulation of undegraded substrate(s) can disrupt other lysosomal degradation processes, vesicular traffic, and lysosomal biogenesis to evoke the diverse pathophysiology that is evident in this complex set of disorders.

  3. 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

  4. Disruption of the Coxsackievirus and Adenovirus Receptor-Homodimeric Interaction Triggers Lipid Microdomain- and Dynamin-dependent Endocytosis and Lysosomal Targeting*

    PubMed Central

    Salinas, Sara; Zussy, Charleine; Loustalot, Fabien; Henaff, Daniel; Menendez, Guillermo; Morton, Penny E.; Parsons, Maddy; Schiavo, Giampietro; Kremer, Eric J.

    2014-01-01

    The coxsackievirus and adenovirus receptor (CAR) serves as a docking factor for some adenovirus (AdV) types and group B coxsackieviruses. Its role in AdV internalization is unclear as studies suggest that its intracellular domain is dispensable for some AdV infection. We previously showed that in motor neurons, AdV induced CAR internalization and co-transport in axons, suggesting that CAR was linked to endocytic and long-range transport machineries. Here, we characterized the mechanisms of CAR endocytosis in neurons and neuronal cells. We found that CAR internalization was lipid microdomain-, actin-, and dynamin-dependent, and subsequently followed by CAR degradation in lysosomes. Moreover, ligands that disrupted the homodimeric CAR interactions in its D1 domains triggered an internalization cascade involving sequences in its intracellular tail. PMID:24273169

  5. 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

  6. 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

  7. Selecting asteroids for a targeted spectroscopic survey

    NASA Astrophysics Data System (ADS)

    Oszkiewicz, D. A.; Kwiatkowski, T.; Tomov, T.; Birlan, M.; Geier, S.; Penttilä, 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

  8. ?-Catenin–dependent lysosomal targeting of internalized tumor necrosis factor-? suppresses caspase-8 activation in apoptosis-resistant colon cancer cells

    PubMed Central

    Han, Jinbo; Sridevi, Priya; Ramirez, Michael; Ludwig, Kirsten J.; Wang, Jean Y. J.

    2013-01-01

    The Wnt/?-catenin pathway is constitutively activated in more than 90% of human colorectal cancer. Activated ?-catenin stimulates cell proliferation and survival, however, its antiapoptotic mechanisms are not fully understood. We show here that activated ?-catenin is required to suppress caspase-8 activation, but only in colon cancer cells that are resistant to tumor necrosis factor-? (TNF)-induced apoptosis. We found that lysosomal delivery of internalized TNF occurred at a faster pace in apoptosis-resistant than in apoptosis-sensitive colon cancer cells. Retardation of endosomal trafficking through vacuolar ATPase (V-ATPase) inhibition enhanced caspase-8 activation in apoptosis-resistant but not apoptosis-sensitive cells. Interestingly, knockdown of ?-catenin also prolonged TNF association with the early endosome and enhanced caspase-8 activation in apoptosis-resistant but not apoptosis-sensitive colon cancer cells. In a mouse model of inflammation-associated colon tumors, we found nuclear expression of ?-catenin, resistance to TNF-induced apoptosis, and reactivation of apoptosis in vivo after cotreatment of TNF with a V-ATPase inhibitor. Together these results suggest that activated ?-catenin can facilitate endosomal trafficking of internalized TNF to suppress caspase-8 activation in colon cancer cells. PMID:23264463

  9. 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.

  10. Target Selection for the LBTI Exozodi Key Science Program

    E-print Network

    Weinberger, Alycia J; 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

    2015-01-01

    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 ~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 ...

  11. Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology - Figure 3

    NSDL National Science Digital Library

    Emma J. Parkinson-Lawrence (South Australian Pathology Services)

    2010-04-01

    This figure shows the morphology of storage compartments commonly observed lysosomal storage disorders: (A) floccular-granular storage, (B) lipid whorls, (C) zebra bodies, and (D) autophagic vacuoles.

  12. Mesoscale nanoparticles selectively target the renal proximal tubule epithelium.

    PubMed

    Williams, Ryan M; Shah, Janki; Ng, Brandon D; Minton, Denise R; Gudas, Lorraine J; Park, Christopher Y; Heller, Daniel A

    2015-04-01

    We synthesized "mesoscale" nanoparticles, approximately 400 nm in diameter, which unexpectedly localized selectively in renal proximal tubules and up to 7 times more efficiently in the kidney than other organs. Although nanoparticles typically localize in the liver and spleen, modulating their size and opsonization potential allowed for stable targeting of the kidneys through a new proposed uptake mechanism. Applying this kidney targeting strategy, we anticipate use in the treatment of renal disease and the study of renal physiology. PMID:25811353

  13. 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

  14. The cell biology of lysosomal storage disorders

    Microsoft Academic Search

    Anthony H. Futerman; Gerrit van Meer

    2004-01-01

    Lysosomal storage disorders, of which more than 40 are known, are caused by the defective activity of lysosomal proteins, which results in the intra-lysosomal accumulation of undegraded metabolites. Despite years of study of the genetic and molecular bases of lysosomal storage disorders, little is known about the events that lead from this intra-lysosomal accumulation to pathology. Here, we summarize the

  15. A targeted multi-enzyme mechanism for selective microtubule

    E-print Network

    Paris-Sud XI, Université de

    1 A targeted multi-enzyme mechanism for selective microtubule polyglutamylation Juliette van Dijk: posttranslational modification, tubulin, motility, polyglutamylase, TTLL Running Title: The multi-enzyme mechanism of polyglutamylation Summary Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths

  16. Selective Tumor Cell Targeting Using Low-Affinity, Multivalent Interactions

    E-print Network

    Kiessling, Laura

    Selective Tumor Cell Targeting Using Low-Affinity, Multivalent Interactions Coby B. Carlson interaction between a cell-binding agent (e.g., monoclonal antibody or fragment thereof) and a tumor- associated antigen to direct a cytotoxic moiety selec- tively to the tumor (2). Despite the potential

  17. A Procedure for Target Market Selection in Tourism

    Microsoft Academic Search

    SooCheong Jang; Alastair M. Morrison; Joseph T. Oleary

    2004-01-01

    The primary objective of this research was to evaluate the attractiveness of travel activity segments to assist with target market selection. Prior studies evaluating segment attractiveness have used ranking systems to determine the best markets. However, due to a lack of precision in these ranking procedures, they have not effectively reflected the degree to which one segment is more attractive

  18. 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

  19. 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.

  20. The management of lysosomal disorders

    Microsoft Academic Search

    James Edmond Wraith

    2004-01-01

    Lysosomal storage disorders (LSDs) are a group of genetic disorders resulting from defective lysosomal metabolism\\/export of a range of naturally occurring compounds. Although individually rare, as a group they constitute a significant burden on society and are expensive to manage.In addition to general palliative care, treatment options for patients with LSDs now include haematopoetic stem cell therapy (HSCT), enzyme replacement

  1. 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

  2. Acoustic gaze adjustments during active target selection in echolocating porpoises.

    PubMed

    Wisniewska, Danuta Maria; Johnson, Mark; Beedholm, Kristian; Wahlberg, Magnus; Madsen, Peter Teglberg

    2012-12-15

    Visually dominant animals use gaze adjustments to organize perceptual inputs for cognitive processing. Thereby they manage the massive sensory load from complex and noisy scenes. Echolocation, as an active sensory system, may provide more opportunities to control such information flow by adjusting the properties of the sound source. However, most studies of toothed whale echolocation have involved stationed animals in static auditory scenes for which dynamic information control is unnecessary. To mimic conditions in the wild, we designed an experiment with captive, free-swimming harbor porpoises tasked with discriminating between two hydrophone-equipped targets and closing in on the selected target; this allowed us to gain insight into how porpoises adjust their acoustic gaze in a multi-target dynamic scene. By means of synchronized cameras, an acoustic tag and on-target hydrophone recordings we demonstrate that porpoises employ both beam direction control and range-dependent changes in output levels and pulse intervals to accommodate their changing spatial relationship with objects of immediate interest. We further show that, when switching attention to another target, porpoises can set their depth of gaze accurately for the new target location. In combination, these observations imply that porpoises exert precise vocal-motor control that is tied to spatial perception akin to visual accommodation. Finally, we demonstrate that at short target ranges porpoises narrow their depth of gaze dramatically by adjusting their output so as to focus on a single target. This suggests that echolocating porpoises switch from a deliberative mode of sensorimotor operation to a reactive mode when they are close to a target. PMID:23175527

  3. Nanoparticle-based biocompatible and long-life marker for lysosome labeling and tracking.

    PubMed

    Shi, Hui; He, Xiaoxiao; Yuan, Yuan; Wang, Kemin; Liu, Dan

    2010-03-15

    In this paper, a novel biocompatible and long-life lysosome labeling and tracking method based on dye entrapped silica nanoparticles (DSiNPs) has been put forward. Through colocalization studies using LysoTracker Green as the standard lysosome marker, it has been demonstrated that DSiNPs selectively accumulated in lysosomes of Hela cells and the photostability of DSiNPs associated with lysosomes was detectable, at least, 30 times as long as that of LysoTracker Green involved in lysosomes. By comparison with LysoTracker Green and Alexa 488-dextran, the fluorescence of DSiNPs could be detected over a 5-day postrecultivation period and the staining pattern in lysosomes could be well retained after cell fixation and permeabilization. In addition, results from MTT assays showed that DSiNPs did not affect the viability of Hela cells at the concentration for lysosome labeling. Primary applications of DSiNPs were then further performed in lysosome tracking in chloroquine-treated Hela cells, and lysosome labeling of differnet cell lines, including MCF-7 cells, MEAR cells, and MSC cells. These results indicated that DSiNPs, therefore, can be used as a biocompatible, long-life, and highly photostable lysosome marker for lysosome-related studies. PMID:20155925

  4. The lysosomal cysteine proteases.

    PubMed

    McGrath, M E

    1999-01-01

    A significant number of exciting papain-like cysteine protease structures have been determined by crystallographic methods over the last several years. This trove of data allows for an analysis of the structural features that empower these molecules as they efficiently carry out their specialized tasks. Although the structure of the paradigm for the family, papain, has been known for twenty years, recent efforts have reaped several structures of specialized mammalian enzymes. This review first covers the commonalities of architecture and purpose of the papain-like cysteine proteases. From that broad platform, each of the lysosomal enzymes for which there is an X-ray structure (or structures) is then examined to gain an understanding of what structural features are used to customize specificity and activity. Structure-based design of inhibitors to control pathological cysteine protease activity will also be addressed. PMID:10410800

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

    Lalonde, Marie-Eve; Cheng, Xue; Côté, 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

  7. Immature and mature species of the human Prostacyclin Receptor are ubiquitinated and targeted to the 26S proteasomal or lysosomal degradation pathways, respectively

    Microsoft Academic Search

    Peter D Donnellan; B Therese Kinsella

    2009-01-01

    BACKGROUND: The human prostacyclin receptor (hIP) undergoes agonist-induced phosphorylation, desensitisation and internalisation and may be recycled to the plasma membrane or targeted for degradation by, as yet, unknown mechanism(s). RESULTS: Herein it was sought to investigate the turnover of the hIP under basal conditions and in response to cicaprost stimulation. It was established that the hIP is subject to low-level

  8. Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation.

    PubMed

    Tacken, Paul J; Ginter, Wiebke; Berod, Luciana; Cruz, Luis J; Joosten, Ben; Sparwasser, Tim; Figdor, Carl G; Cambi, Alessandra

    2011-10-13

    Targeting antigens to dendritic cell (DC)-specific receptors, such as DC-SIGN, induces potent T cell-mediated immune responses. DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD). Thus far, only antibodies binding the CRD have been used to target antigens to DC-SIGN. We evaluated the endocytic pathway triggered by antineck antibodies as well as their intracellular routing and ability to induce CD8(+) T-cell activation. In contrast to anti-CRD antibodies, antineck antibodies induced a clathrin-independent mode of DC-SIGN internalization, as demonstrated by the lack of colocalization with clathrin and the observation that silencing clathrin did not affect antibody internalization in human DCs. Interestingly, we observed that anti-neck and anti-CRD antibodies were differentially routed within DCs. Whereas anti-CRD antibodies were mainly routed to late endosomal compartments, anti-neck antibodies remained associated with early endosomal compartments positive for EEA-1 and MHC class I for up to 2 hours after internalization. Finally, cross-presentation of protein antigen conjugated to antineck antibodies was approximately 1000-fold more effective than nonconjugated antigen. Our studies demonstrate that anti-neck antibodies trigger a distinct mode of DC-SIGN internalization that shows potential for targeted vaccination strategies. PMID:21860028

  9. 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.

  10. 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.; López-Sánchez, Á. 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?.

  11. Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology - Figure 2

    NSDL National Science Digital Library

    Emma J. Parkinson-Lawrence (South Australian Pathology Services)

    2010-04-01

    This figure is a timeline of discoveries in lysosomal storage disorders and their impact on cell biology, including endocytic processes and the subsequent development of enzyme replacement therapy (ERT).

  12. Target Inhibition Networks: Predicting Selective Combinations of Druggable Targets to Block Cancer Survival Pathways

    PubMed Central

    Tang, Jing; Karhinen, Leena; Xu, Tao; Szwajda, Agnieszka; Yadav, Bhagwan; Wennerberg, Krister; Aittokallio, Tero

    2013-01-01

    A recent trend in drug development is to identify drug combinations or multi-target agents that effectively modify multiple nodes of disease-associated networks. Such polypharmacological effects may reduce the risk of emerging drug resistance by means of attacking the disease networks through synergistic and synthetic lethal interactions. However, due to the exponentially increasing number of potential drug and target combinations, systematic approaches are needed for prioritizing the most potent multi-target alternatives on a global network level. We took a functional systems pharmacology approach toward the identification of selective target combinations for specific cancer cells by combining large-scale screening data on drug treatment efficacies and drug-target binding affinities. Our model-based prediction approach, named TIMMA, takes advantage of the polypharmacological effects of drugs and infers combinatorial drug efficacies through system-level target inhibition networks. Case studies in MCF-7 and MDA-MB-231 breast cancer and BxPC-3 pancreatic cancer cells demonstrated how the target inhibition modeling allows systematic exploration of functional interactions between drugs and their targets to maximally inhibit multiple survival pathways in a given cancer type. The TIMMA prediction results were experimentally validated by means of systematic siRNA-mediated silencing of the selected targets and their pairwise combinations, showing increased ability to identify not only such druggable kinase targets that are essential for cancer survival either individually or in combination, but also synergistic interactions indicative of non-additive drug efficacies. These system-level analyses were enabled by a novel model construction method utilizing maximization and minimization rules, as well as a model selection algorithm based on sequential forward floating search. Compared with an existing computational solution, TIMMA showed both enhanced prediction accuracies in cross validation as well as significant reduction in computation times. Such cost-effective computational-experimental design strategies have the potential to greatly speed-up the drug testing efforts by prioritizing those interventions and interactions warranting further study in individual cancer cases. PMID:24068907

  13. 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

    2015-04-01

    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

  14. Lysosomal Enzymes and their Receptors in Invertebrates: An Evolutionary Perspective.

    PubMed

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

    Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here. PMID:25692847

  15. Selective Genomic Targeting by FRA-2/FOSL2 Transcription Factor

    PubMed Central

    Davies, Jeff S.; Klein, David C.; Carter, David A.

    2011-01-01

    FRA-2/FOSL2 is a basic region-leucine zipper motif transcription factor that is widely expressed in mammalian tissues. The functional repertoire of this factor is unclear, partly due to a lack of knowledge of genomic sequences that are targeted. Here, we identified novel, functional FRA-2 targets across the genome through expression profile analysis in a knockdown transgenic rat. In this model, a nocturnal rhythm of pineal gland FRA-2 is suppressed by a genetically encoded, dominant negative mutant protein. Bioinformatic analysis of validated sets of FRA-2-regulated and -nonregulated genes revealed that the FRA-2 regulon is limited by genomic target selection rules that, in general, transcend core cis-sequence identity. However, one variant AP-1-related (AP-1R) sequence was common to a subset of regulated genes. The functional activity and protein binding partners of a candidate AP-1R sequence were determined for a novel FRA-2-repressed gene, Rgs4. FRA-2 protein preferentially associated with a proximal Rgs4 AP-1R sequence as demonstrated by ex vivo ChIP and in vitro EMSA analysis; moreover, transcriptional repression was blocked by mutation of the AP-1R sequence, whereas mutation of an upstream consensus AP-1 family sequence did not affect Rgs4 expression. Nocturnal changes in protein complexes at the Rgs4 AP-1R sequence are associated with FRA-2-dependent dismissal of the co-activator, CBP; this provides a mechanistic basis for Rgs4 gene repression. These studies have also provided functional insight into selective genomic targeting by FRA-2, highlighting discordance between predicted and actual targets. Future studies should address FRA-2-Rgs4 interactions in other systems, including the brain, where FRA-2 function is poorly understood. PMID:21367864

  16. 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

  17. The Endosomal Sorting Complex Required for Transport Pathway Mediates Chemokine Receptor CXCR4-promoted Lysosomal Degradation of the Mammalian Target of Rapamycin Antagonist DEPTOR.

    PubMed

    Verma, Rita; Marchese, Adriano

    2015-03-13

    G protein-coupled receptor (GPCR) signaling mediates many cellular functions, including cell survival, proliferation, and cell motility. Many of these processes are mediated by GPCR-promoted activation of Akt signaling by mammalian target of rapamycin complex 2 (mTORC2) and the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase 1 (PDK1) pathway. However, the molecular mechanisms by which GPCRs govern Akt activation by these kinases remain poorly understood. Here, we show that the endosomal sorting complex required for transport (ESCRT) pathway mediates Akt signaling promoted by the chemokine receptor CXCR4. Pharmacological inhibition of heterotrimeric G protein G?i or PI3K signaling and siRNA targeting ESCRTs blocks CXCR4-promoted degradation of DEPTOR, an endogenous antagonist of mTORC2 activity. Depletion of ESCRTs by siRNA leads to increased levels of DEPTOR and attenuated CXCR4-promoted Akt activation and signaling, consistent with decreased mTORC2 activity. In addition, ESCRTs likely have a broad role in Akt signaling because ESCRT depletion also attenuates receptor tyrosine kinase-promoted Akt activation and signaling. Our data reveal a novel role for the ESCRT pathway in promoting intracellular signaling, which may begin to identify the signal transduction pathways that are important in the physiological roles of ESCRTs and Akt. PMID:25605718

  18. Pathomechanisms in Lysosomal Storage Disorders

    PubMed Central

    Walkley, Steven U.; Vanier, Marie T.

    2015-01-01

    Lysosomal diseases are inherited metabolic disorders caused by defects in a wide spectrum of lysosomal and a few non-lysosomal proteins. In most cases a single type of primary storage material is identified, which has been used to name and classify the disorders: hence the terms sphingolipidoses, gangliosidoses, mucopolysaccharidoses, glycoproteinoses, and so forth. In addition to this primary storage, however, a host of secondary storage products can also be identified, more often than not having no direct link to the primary protein defect. Lipids - glycosphingolipids and phospholipids, as well as cholesterol - are the most ubiquitous and best studied of these secondary storage materials. While in the past typically considered nonspecific and nonconsequential features of these diseases, newer studies suggest direct links between secondary storage and disease pathogenesis and support the view that understanding all aspects of this sequestration process will provide important insights into the cell biology and treatment of lysosomal disease. PMID:19111580

  19. The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid

    PubMed Central

    2014-01-01

    Background Alzheimer’s disease (AD) is characterized by cerebral deposition of ?-amyloid peptide (A?). A? is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by ?- and ?-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate A? production, although the exact organelle in which A? is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating A? production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing. Results We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because ?-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective ?-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces A? production by more than a third. Conclusion These data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of A? production. PMID:25085554

  20. [Current therapeutic strategies in lysosomal disorders].

    PubMed

    Kaminsky, Pierre; Lidove, Olivier

    2014-11-01

    The lysosomal storage disorders (LSD) comprise a heterogeneous group of inborn errors of metabolism. The resulting enzymatic defect leads to accumulation of its substrate in the lysosome. Their clinical patterns reflect the site of substrate storage. Central nervous system involvement is often present in the younger patients affected by the most severe phenotypes. Substantial progress has been made in the pathophysiological knowledge, leading to new therapeutic options in LSD. Enzyme replacement therapy (ERT) is the dominant approach and is actually proposed in six LSD: Gaucher disease, Fabry disease, Pompe disease and mucopolysaccharidoisis (MPS) I (Hurler disease), II (Hunter disease) and VI (Maroteaux-Lamy disease). This treatment reduces lysosomal storage, and sometimes reduces, but most often limits the progression of visceral involvement and of its clinical consequences. However, ERT does not cross the blood-brain barrier and is ineffective on neurological symptoms. In the younger patients with MPS I (Hurler disease) and with selected cases of other LSD, haematopoietic stem cell transplantation remains the optimal option. Other strategies using small molecules are being explored in order to cross the blood-brain barrier. This includes substrate reduction or depletion therapies, which decrease the amount of substrate, and the use of pharmacological chaperones, which enhance the residual activity of the mutant enzyme. Miglustat is the proposed substrate reduction therapy in Niemann-Pick C disease and clinical trials are actually performed in several LSD using other substrate reduction or chaperone drugs. PMID:24863660

  1. 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

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

    SciTech Connect

    Bertozzi, C.R. [Univ. of California, Berkeley, CA (United States); [Lawrence Berkeley National Lab., CA (United States)

    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.

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

    SciTech Connect

    Batalha, Natalie M. [Department of Physics and Astronomy, San Jose State University, San Jose, CA 95192 (United States); Borucki, William J.; Koch, David G.; Bryson, Stephen T.; Haas, Michael R. [NASA Ames Research Center, Moffett Field, CA (United States); Brown, Timothy M. [Las Cumbres Observatory Global Observatory Telescope Network, Goleta, CA 93117 (United States); Caldwell, Douglas A. [SETI Institute, Mountain View, CA 94043 (United States); Hall, Jennifer R. [Orbital Sciences Corporation/NASA Ames Research Center, Moffett Field, CA 94035 (United States); Gilliland, Ronald L. [STScI, Baltimore, MD 21218 (United States); Latham, David W.; Meibom, Soren [Harvard-Smithsonian, CfA, Cambridge, MA 02138 (United States); Monet, David G. [U.S. Naval Observatory, Flagstaff, AZ 86001 (United States)], E-mail: Natalie.Batalha@sjsu.edu

    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.

  4. Therapy of Lysosomal Storage Diseases

    Microsoft Academic Search

    Ulrich Matzner

    Lysosomes are membrane-surrounded organelles which are present in all nucleated mammalian cells. They function to degrade\\u000a both intra- and extracellular macromolecules to low molecular components that are transported to the cytoplasm for reutilization\\u000a in the biosynthetic pathways of the cell. Up to now more than 50 different lysosomal hydrolases catalyzing the degradation\\u000a of proteins, lipids, nucleic acids and carbohydrates have

  5. 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

  6. 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

  7. 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.

  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. Reporter assay for endo/lysosomal escape of toxin-based therapeutics.

    PubMed

    Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F; Fuchs, Hendrik; Weng, Alexander

    2014-05-01

    Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM. PMID:24859158

  11. Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

    PubMed Central

    Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F.; Fuchs, Hendrik; Weng, Alexander

    2014-01-01

    Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM. PMID:24859158

  12. Target selection and development in 3D laser scanning based on sampling interval

    Microsoft Academic Search

    Guigang Shi; Jianbo Tang; Yunlan Guan; Xiaojun Cheng

    2010-01-01

    In 3D laser scanning technology, it is very important for targets to the point cloud registration. Because of the difference of noise, target types and target materials, the difficult extent is different to extract target centre, even the target centre could not be extracted, which affects eventually point cloud registration and its accuracy. This requires scanning job must select rightly

  13. Lysosome-mediated processing of chromatin in senescence.

    PubMed

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M; Rai, Taranjit Singh; Shah, Parisha P; Hewitt, Graeme; Korolchuk, Viktor I; Passos, Joao F; Wu, Hong; Berger, Shelley L; Adams, Peter D

    2013-07-01

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly ?-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression. PMID:23816621

  14. Lysosome-mediated processing of chromatin in senescence

    PubMed Central

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M.; Rai, Taranjit Singh; Shah, Parisha P.; Hewitt, Graeme; Korolchuk, Viktor I.; Passos, Joao F.; Wu, Hong; Berger, Shelley L.

    2013-01-01

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly ?-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression. PMID:23816621

  15. Lysosomal membrane permeabilization in cell death: concepts and challenges.

    PubMed

    Repnik, Urška; Hafner ?esen, Maruša; Turk, Boris

    2014-11-01

    Late endocytic compartments include late endosomes, lysosomes and hybrid organelles. In the acidic lumen, cargo material derived from endocytosed and phagocytosed extracellular material and autophagy-derived intracellular material is degraded. In the event of lysosomal membrane permeabilization (LMP), the function of endo/lysosomal compartment is affected and the luminal contents are released into the cytosol to various extents. LMP can be a result of osmotic lysis or direct membranolytic activity of the compounds that accumulate in the lumen of endo/lysosomes. In addition to several synthetic compounds, such as dipeptide methyl esters and lysosomotropic detergents, endogenous agents that can cause LMP include ROS and lipid metabolites such as sphingosine and phosphatidic acid. Depending on the cell type and the dose, LMP can initiate the lysosomal apoptotic pathway, pyroptosis or necrosis. LMP can also amplify cell death signaling that was initiated outside the endocytic compartment, and hamper cell recovery via autophagy. However, mechanisms that connect LMP with cell death signaling are poorly understood, with the exception of the proteolytic activation of Bid by aspartic cathepsin D and cysteine cathepsins. Determination of LMP in a cell model system is methodologically challenging. Even more difficult is to prove that LMP is the primary event leading to cell death. Nevertheless, LMP may prove to be a valuable approach in therapy, either as a trigger of cell death or as a mechanism of therapeutic drug release in the case of delivery systems that target the endocytic pathway. PMID:24984038

  16. Cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic Beta-cell apoptosis in high glucose.

    PubMed

    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

  17. 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.

  18. 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

  19. Immunochemistry of Lysosomal Storage Disorders

    Microsoft Academic Search

    Emma Parkinson-Lawrence; Maria Fuller; John J. Hopwood; Peter J. Meikle; Doug A. Brooks

    2006-01-01

    Background: Lysosomal storage disorders are a group of genetic diseases, each with a broad spectrum of clinical presentation that ranges from attenuated to severe. The immunochemical analysis of patient samples is aimed at several key aspects of patient management, including early detection of the disorder, prediction of clinical severity, determining the most appropriate therapeutic regimen, and monitoring of patients on

  20. Rescue of compromised lysosomes enhances degradation of photoreceptor outer segments and reduce lipofuscin-like autofluorescence

    PubMed Central

    Guha, Sonia; Liu, Ji; Baltazar, Gabe; Laties, Alan M.; Mitchell, Claire H.

    2014-01-01

    Healthful cell maintenance requires the efficient degradative processing and removal of waste material. Retinal pigmented epithelial (RPE) cells have the onerous task of degrading both internal cellular debris generated through autophagy as well as phagocytosed photoreceptor outer segments. We propose that the inadequate processing material with the resulting accumulation of cellular waste contributes to the downstream pathologies characterized as age-related macular degeneration (AMD). The lysosomal enzymes responsible for clearance function optimally over a narrow range of acidic pH values; elevation of lysosomal pH by compounds like chloroquine or A2E can impair degradative enzyme activity and lead to a lipofuscin-like autofluorescence. Restoring acidity to the lysosomes of RPE cells can enhance activity of multiple degradative enzymes and is therefore a logical target in early AMD. We have identified several approaches to reacidify lysosomes of compromised RPE cells; stimulation of beta-adrenergic, A2A adenosine and D5 dopamine receptors each lowers lysosomal pH and improves degradation of photoreceptor outer segments. Activation of the CFTR chloride channel also reacidifies lysosomes and increases degradation. These approaches also restore the lysosomal pH of RPE cells from aged ABCA4?/? mice with chronically high levels of A2E, suggesting that functional signaling pathways to reacidify lysosomes are retained in aged cells like those in patients with AMD. Acidic nanoparticles transported to RPE lysosomes also lower pH and improve degradation of outer segments. In summary, the ability of diverse approaches to lower lysosomal pH and enhance outer segment degradation support the proposal that lysosomal acidification can prevent the accumulation of lipofuscin-like material in RPE cells. PMID:24664687

  1. Lysosomal Phospholipase A2 and Phospholipidosis†

    PubMed Central

    Hiraoka, Miki; Abe, Akira; Lu, Ye; Yang, Kui; Han, Xianlin; Gross, Richard W.; Shayman, James A.

    2006-01-01

    A lysosomal phospholipase A2, LPLA2, was recently characterized and shown to have substrate specificity for phosphatidylcholine and phosphatidylethanolamine. LPLA2 is ubiquitously expressed but is most highly expressed in alveolar macrophages. Double conditional gene targeting was employed to elucidate the function of LPLA2. LPLA2-deficient mice (Lpla2?/?) were generated by the systemic deletion of exon 5 of the Lpla2 gene, which encodes the lipase motif essential for the phospholipase A2 activity. The survival of the Lpla2?/? mice was normal. Lpla2?/? mouse mating pairs yielded normal litter sizes, indicating that the gene deficiency did not impair fertility or fecundity. Alveolar macrophages from wild-type but not Lpla2?/? mice readily degraded radiolabeled phosphatidylcholine. A marked accumulation of phospholipids, in particular phosphatidylethanolamine and phosphatidylcholine, was found in the alveolar macrophages, the peritoneal macrophages, and the spleens of Lpla2?/? mice. By 1 year of age, Lpla2?/? mice demonstrated marked splenomegaly and increased lung surfactant phospholipid levels. Ultrastructural examination of Lpla2?/? mouse alveolar and peritoneal macrophages revealed the appearance of foam cells with lamellar inclusion bodies, a hallmark of cellular phospholipidosis. Thus, a deficiency of lysosomal phospholipase A2 results in foam cell formation, surfactant lipid accumulation, splenomegaly, and phospholipidosis in mice. PMID:16880524

  2. 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

  3. Saccade-target selection of dyslexic children when reading Chinese

    PubMed Central

    Pan, Jinger; Yan, Ming; Laubrock, Jochen; Shu, Hua; Kliegl, Reinhold

    2015-01-01

    This study investigates the eye movements of dyslexic children and their age-matched controls when reading Chinese. Dyslexic children exhibited more and longer fixations than age-matched control children, and an increase of word length resulted in a greater increase in the number of fixations and gaze durations for the dyslexic than for the control readers. The report focuses on the finding that there was a significant difference between the two groups in the fixation landing position as a function of word length in single-fixation cases, while there was no such difference in the initial fixation of multi-fixation cases. We also found that both groups had longer incoming saccade amplitudes while the launch sites were closer to the word in single fixation cases than in multi-fixation cases. Our results suggest that dyslexic children's inefficient lexical processing, in combination with the absence of orthographic word boundaries in Chinese, leads them to select saccade targets at the beginning of words conservatively. These findings provide further evidence for parafoveal word segmentation during reading of Chinese sentences. PMID:24508073

  4. Brief exposure to copper activates lysosomal exocytosis.

    PubMed

    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

    Copper (Cu) is essential mineral, but its toxicity necessitates existence of powerful machinery responsible for the extraction of excess Cu from the cell. Cu exposure was recently shown to induce the translocation of Cu pump ATP7B to the lysosomes followed by lysosomal exocytosis. Here we sought to investigate the mechanisms underlying the effect of Cu on lysosomal exocytosis. We found that brief exposure to Cu activates lysosomal exocytosis, which was measured as a release of the lysosomal digestive enzyme ?-hexosaminidase (?-hex) into the extracellular medium and by the presence lysosomal protein LAMP1 at the plasma membrane. Such release depends on calcium (Ca) and on the lysosomal SNARE VAMP7. ATP7B knockdown using RNAi suppressed the basal lysosomal exocytosis, but did not affect the ability of Cu to activate it. ATP7B knockdown was associated with sustained oxidative stress. The removal of Ca from the extracellular medium suppressed the Cu-dependent component of the lysosomal exocytosis. We propose that Cu promotes lysosomal exocytosis by facilitating a Ca-dependent step of the lysosomal exocytosis. PMID:25620123

  5. Measuring relative lysosomal volume for monitoring lysosomal storage diseases.

    PubMed

    Te Vruchte, Danielle; Wallom, Kerri L; Platt, Frances M

    2015-01-01

    Biomarkers are important tools in medicine, which can be used for monitoring disease progression and response to therapy. One of the main problems in rare lysosomal storage diseases is that there are over 70 different diseases, all with different biochemical storage profiles. Developing biochemical biomarkers therefore requires an individual assay per disease/subgroup of diseases. An alternative approach is to develop an assay that is independent of the specific macromolecules stored. This chapter discusses an assay that may serve as a universal biomarker for these diseases and measures the expansion of the late endosomal/lysosomal system. We have developed an assay that takes advantage of a commercially available late endosomal/lysosomal probe, LysoTracker, which becomes trapped in the acidic compartment of cells and emits a fluorescent signal that can be detected using flow cytometry. In this chapter, we detail the methodology behind this assay and discuss the factors that need to be considered when establishing this assay in clinical and research settings. PMID:25665453

  6. 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

  7. Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

    PubMed Central

    Liu, Bo; Fang, Mengdie; Hu, Ye; Huang, Baoshan; Li, Ning; Chang, Chunmei; Huang, Rui; Xu, Xiao; Yang, Zhenggang; Chen, Zhi; Liu, Wei

    2014-01-01

    Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC. PMID:24401568

  8. 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

  9. 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)kgday(-1) and 0.47 (0.03)kgday(-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

  10. Inhibitor screening of pharmacological chaperones for lysosomal ?-glucocerebrosidase by capillary electrophoresis

    Microsoft Academic Search

    Meera Shanmuganathan; Philip Britz-McKibbin

    2011-01-01

    Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based\\u000a on enhanced stabilization and trafficking of mutant protein upon orthosteric and\\/or allosteric binding. Herein, we introduce\\u000a a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal\\u000a enzyme, ?-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the

  11. Studies on the impact of amine-containing compounds on lysosomes

    E-print Network

    Funk, Ryan Sol

    2011-12-31

    ………………………………………………. 98 xii 3.3. Results and discussion………………………………………………………………..… 99 3.3.1. Effect of pharmacologically unrelated CADs on the cellular accumulation of substrates for ion trapping in lysosomes………………………. 100 3.3.2. Importance of intact... to the pharmacological and toxicological effects elicited by many of these drugs. Also, the potential that these off-target effects can impact the ability of lysosomes to contribute to 5 the tissue uptake and accumulation of drugs reveals the possibility that one...

  12. Target Tracking with Online Feature Selection in FLIR Imagery Vijay Venkataraman, Guoliang Fan and Xin Fan

    E-print Network

    Fan, Guoliang

    Target Tracking with Online Feature Selection in FLIR Imagery Vijay Venkataraman, Guoliang Fan tracking algo- rithm for FLIR imagery. A dual foreground and background model is proposed for target that the proposed algorithm can accurately track poorly-visible targets in FLIR imagery even with strong ego

  13. Adverse Selection in Acquisitions of Small Manufacturing Firms: A Comparison of Private and Public Targets

    Microsoft Academic Search

    Jung-Chin Shen; Jeffrey J. Reuer

    2005-01-01

    This study investigates acquisitions of small manufacturing firms and compares private and public targets. We develop the argument that private targets tend to involve higher transaction costs in the presence of adverse selection problems than their public counterparts. Consistent with predictions, the empirical evidence indicates that bidders choose to acquire public rather than private targets when acquiring young firms and

  14. Immune system irregularities in lysosomal storage disorders

    Microsoft Academic Search

    Julian A. Castaneda; Ming J. Lim; Jonathan D. Cooper; David A. Pearce

    2008-01-01

    Lysosomal storage disorders (LSDs) are genetically inherited diseases characterized by the accumulation of disease-specific\\u000a biological materials such as proteolipids or metabolic intermediates within the lysosome. The lysosomal compartment’s central\\u000a importance to normal cellular function can be appreciated by examining the various pathologies that arise in LSDs. These disorders\\u000a are invariably fatal, and many display profound neurological impairment that begins in

  15. Non-esterified Cholesterol Content of Lysosomes Modulates Susceptibility to Oxidant-induced Permeabilization

    PubMed Central

    Reiners, John J.; Kleinman, Miriam; Kessel, David; Mathieu, Patricia A.; Caruso, Joseph A.

    2010-01-01

    Reactive oxygen species (ROS) can induce lysosomal membrane permeabilization (LMP). Photoirradiation of murine hepatoma 1c1c7 cultures preloaded with the photosensitizer NPe6 generates singlet oxygen within acidic organelles, and causes LMP and the activation of procaspases. Treatment with the cationic amphiphilic drugs (CADs) U18666A, imipramine, and clozapine stimulated the accumulation of filipin-stainable non-esterified cholesterol/sterols in late endosomes/lysosomes, but not in mitochondria. Concentration-response studies demonstrated an inverse relationship between lysosomal non-esterified cholesterol/sterol contents and susceptibility to NPe6 photoirradiation-induced intracellular membrane oxidation, LMP, and activation of procaspases-9 and -3. Similarly, the kinetics of restoration of NPe6 photoirradiation-induced LMP paralleled the losses of lysosomal cholesterol that occurred upon replating U18666A-treated cultures in CAD-free medium. Consistent with the oxidation of lysosomal cholesterol, filipin staining in U18666A-treated cultures progressively decreased with increasing photoirradiating light dose. U18666A also suppressed the inductions of LMP and procaspase activation by exogenously added hydrogen peroxide. However, neither U18666A nor imipramine suppressed the induction of apoptosis by agents that did not directly induce LMP. These studies indicate that lysosomal non-esterified cholesterol/sterol content modulates susceptibility to ROS-induced LMP, and possibly does so by being an alternative target for oxidants and lowering the probability of damage to other lysosomal membrane lipids and/or proteins. PMID:21074609

  16. Subversion of a lysosomal pathway regulating neutrophil apoptosis by a major bacterial toxin, pyocyanin.

    PubMed

    Prince, Lynne R; Bianchi, Stephen M; Vaughan, Kathryn M; Bewley, Martin A; Marriott, Helen M; Walmsley, Sarah R; Taylor, Graham W; Buttle, David J; Sabroe, Ian; Dockrell, David H; Whyte, Moira K B

    2008-03-01

    Neutrophils undergo rapid constitutive apoptosis that is accelerated following bacterial ingestion as part of effective immunity, but is also accelerated by bacterial exotoxins as a mechanism of immune evasion. The paradigm of pathogen-driven neutrophil apoptosis is exemplified by the Pseudomonas aeruginosa toxic metabolite, pyocyanin. We previously showed pyocyanin dramatically accelerates neutrophil apoptosis both in vitro and in vivo, impairs host defenses, and favors bacterial persistence. In this study, we investigated the mechanisms of pyocyanin-induced neutrophil apoptosis. Pyocyanin induced early lysosomal dysfunction, shown by altered lysosomal pH, within 15 min of exposure. Lysosomal disruption was followed by mitochondrial membrane permeabilization, caspase activation, and destabilization of Mcl-1. Pharmacological inhibitors of a lysosomal protease, cathepsin D (CTSD), abrogated pyocyanin-induced apoptosis, and translocation of CTSD to the cytosol followed pyocyanin treatment and lysosomal disruption. A stable analog of cAMP (dibutyryl cAMP) impeded the translocation of CTSD and prevented the destabilization of Mcl-1 by pyocyanin. Thus, pyocyanin activated a coordinated series of events dependent upon lysosomal dysfunction and protease release, the first description of a bacterial toxin using a lysosomal cell death pathway. This may be a pathological pathway of cell death to which neutrophils are particularly susceptible, and could be therapeutically targeted to limit neutrophil death and preserve host responses. PMID:18292577

  17. 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

  18. 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

  19. Docosahexaenoic acid selectively inhibits plasma membrane targeting of lipidated proteins

    Microsoft Academic Search

    Jeongmin Seo; Rola Barhoumi; Arthur E. Johnson; Joanne R. Lupton; Robert S. Chapkin

    2006-01-01

    Membrane localization of lipidated cytosolic signaling proteins is mediated by interactions between specific lipid anchors and membranes, but little is known about the regulatory role of membrane composition in lipidated protein membrane targeting. Here, using green fluorescent protein (GFP) chimeras and quantitative fluorescence microscopy in living mouse colonocytes, we show that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid (PUFA)

  20. Time Series Analysis of Inflation Targeting in Selected Countries

    Microsoft Academic Search

    Ismail H. Genc; Minsoo Lee; Candelaria O. Rodríguez; Zachary Lutz

    2007-01-01

    We analyze a set of countries which adopted inflation targeting (IT) as a policy tool. We model the pre?IT period with ARMA and GARCH methods, and conduct the one?step ahead forecasting for the remainder of the times series data. The actual and forecasted inflation levels are compared for each country. We find that even though the actual inflation levels are

  1. MECHANISMS OF LYSOSOMAL ENZYME RELEASE FROM HUMAN LEUKOCYTES

    PubMed Central

    Zurier, Robert B.; Hoffstein, Sylvia; Weissmann, Gerald

    1973-01-01

    In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (?-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10-3–10-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10-3 M cyclic nucleotides and 2.8 x 10-4–2.8 x 10-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE1 also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules. PMID:4125373

  2. Lysosomal cysteine proteases: more than scavengers

    Microsoft Academic Search

    Boris Turk; Dušan Turk; Vito Turk

    2000-01-01

    Lysosomal cysteine proteases were believed to be mainly involved in intracellular protein degradation. Under special conditions they have been found outside lysosomes resulting in pathological conditions. With the discovery of a series of new cathepsins with restricted tissue distributions, it has become evident that these enzymes must be involved in a range of specific cellular tasks much broader than as

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

    PubMed

    Maschewsky-Schneider, U; Goecke, M; Gocke, M; Hölscher, U; Kolip, P; Kuhn, A; Sewöster, 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

  4. 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

  5. A key enzyme in the biogenesis of lysosomes is a protease that regulates cholesterol metabolism.

    PubMed

    Marschner, Katrin; Kollmann, Katrin; Schweizer, Michaela; Braulke, Thomas; Pohl, Sandra

    2011-07-01

    Mucolipidosis II is a severe lysosomal storage disorder caused by defects in the ? and ? subunits of the hexameric N-acetylglucosamine-1-phosphotransferase complex essential for the formation of the mannose 6-phosphate targeting signal on lysosomal enzymes. Cleavage of the membrane-bound ?/?-subunit precursor by an unknown protease is required for catalytic activity. Here we found that the ?/?-subunit precursor is cleaved by the site-1 protease (S1P) that activates sterol regulatory element-binding proteins in response to cholesterol deprivation. S1P-deficient cells failed to activate the ?/?-subunit precursor and exhibited a mucolipidosis II-like phenotype. Thus, S1P functions in the biogenesis of lysosomes, and lipid-independent phenotypes of S1P deficiency may be caused by lysosomal dysfunction. PMID:21719679

  6. 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

  7. Kinases and glutathione transferases: selective and sensitive targeting

    Microsoft Academic Search

    Yasemin G. Isgor; Belgin S. Isgor

    2011-01-01

    Kinases, representing almost 500 proteins in the human genome, are responsible for catalyzing the phosphorylation reaction\\u000a of amino acid residues at their targets. As the largest family of kinases, the protein tyrosine kinases (PTKs) have roles\\u000a in controlling the essential cellular activities, and their deregulation is generally related to pathologic conditions. The\\u000a recent efforts on identifying their signal transducer or

  8. 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

  9. Selective Bacteriophage Screening Targeting GqQ209L Protein Abdul Karim Khan1

    E-print Network

    Zhou, Yaoqi

    Selective Bacteriophage Screening Targeting GqQ209L Protein Abdul Karim Khan1 , Kamakshi Sishtila1 melanoma in majority of all oncogenic cases. The method of bacteriophage display was used to find a peptide

  10. 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.

  11. LYSOSOMES OF THE ARTERIAL WALL

    PubMed Central

    Peters, T. J.; Müller, M.; de Duve, Amo C.

    1972-01-01

    Smooth muscle cells were dissociated from normal rabbit aorta by incubating the tissue in Hanks' solution containing elastase, collagenase, and hyaluronidase. The isolated cells contained significant amounts of the following acid hydrolases: N-acetyl-?-glucosaminidase, N-acetyl-?-galactosaminidase, ?-galactosidase, ?-glucuronidase, ?-mannosidase, ?-glucosidase, acid phosphatase, and cathepsins C and D. The cells were disrupted and fractionated by isopycnic centrifugation on sucrose density gradients in the Beaufay automatic zonal rotor. Lysosomes with a modal density of 1.16 were identified by the distribution of these acid hydrolases and by the latency of N-acetyl-?-glucosaminidase and ?-galactosidase. Other particulate enzymes studied in these sucrose gradients included cytochrome oxidase and monoamine oxidase (mitochondria), 5'-nucleotidase and leucyl-?-naphthylamidase (plasma membrane), and catalase (? peroxisome). This microanalytical subcellular fractionation technique is applicable to the study of milligram quantities of many other tissues, both normal and pathological. PMID:4343242

  12. von Economo neurones are selectively targeted in frontotemporal dementia

    PubMed Central

    Santillo, A F; Nilsson, C; Englund, E

    2013-01-01

    Background von Economo neurones (VEN) are bipolar neurones located in the anterior cingulate cortex (ACC) and the frontoinsular cortex (FI), areas affected early in behavioural variant frontotemporal dementia (bvFTD), in which VEN may constitute a selectively vulnerable cellular population. Aim A previous study has shown a selective loss of VEN in FTD above other neurones in the ACC of FTD. The aim of this study was to confirm this finding in a larger cohort, using a different methodology, and to examine VEN loss in relation to neuropathological severity and molecular pathology. Methods VEN and neighbouring neurones (NN) were quantified in layers Va and Vb of the right dorsal ACC in 21 cases of bvFTD, 10 cases of Alzheimer's disease (AD) and 10 non-demented controls (NDC). Results A marked VEN reduction was seen in all FTD cases. In the neuropathologically early cases of FTD (n = 13), VEN/10 000 NN was significantly reduced by 53% compared with NDC (P < 0.001) and 41% compared with AD (P = 0.019), whereas AD patients showed a non-significant 30% reduction of VEN/10 000 NN compared with NDC. VEN reduction was present in all protein pathology subgroups. Discussion In conclusion, this study confirms selective sensitivity of VEN in FTD and suggests that VEN loss is an early event in the neurodegenerative process. PMID:23346995

  13. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle

    PubMed Central

    Schiffelers, Raymond M.; Ansari, Aslam; Xu, Jun; Zhou, Qin; Tang, Qingquan; Storm, Gert; Molema, Grietje; Lu, Patrick Y.; Scaria, Puthupparampil V.; Woodle, Martin C.

    2004-01-01

    Potent sequence selective gene inhibition by siRNA ‘targeted’ therapeutics promises the ultimate level of specificity, but siRNA therapeutics is hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these problems, ligand-targeted, sterically stabilized nanoparticles have been adapted for siRNA. Self-assembling nanoparticles with siRNA were constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis. Cell delivery and activity of PEGylated PEI was found to be siRNA sequence specific and depend on the presence of peptide ligand and could be competed by free peptide. Intravenous administration into tumor-bearing mice gave selective tumor uptake, siRNA sequence-specific inhibition of protein expression within the tumor and inhibition of both tumor angiogenesis and growth rate. The results suggest achievement of two levels of targeting: tumor tissue selective delivery via the nanoparticle ligand and gene pathway selectivity via the siRNA oligonucleotide. This opens the door for better targeted therapeutics with both tissue and gene selectivity, also to improve targeted therapies with less than ideal therapeutic targets. PMID:15520458

  14. Improved band similarity-based hyperspectral imagery band selection for target detection

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Cao, Yan; Zhuo, Li; Wang, Chao; Zhou, Qianlan

    2015-01-01

    The high dimensionality of hyperspectral imagery is a huge challenge for remote sensing data processing. Band selection utilizes the most distinctive and informative band subset to reduce data dimensions. Although band selection can significantly alleviate the computational burden, the process itself may be time consuming because it needs to take all pixels into consideration, especially when the image spatial size is larger. An improved band similarity-based band selection method is proposed for hyperspectral imagery target detection, which includes four steps: (1) bad bands are removed by data preprocessing; (2) several selected pixels are used for band selection instead of using all the pixels to reduce the computational complexity; (3) hyperspectral imagery is analyzed for target detection; and (4) the number of selected bands is determined by adjusting the threshold of similarity metric, to ensure target detection operators have the best performance with selected bands. In the example, the well-known adaptive coherence estimator detector was used to evaluate the effectiveness of the proposed band selection method. The receiver operating characteristics curves were plotted to prove the proposed algorithm quantitatively. The experimental results show that our method can yield a better result in target detection than other band selection methods.

  15. Lysosomal Destabilization Contributes to Apoptosis of Germinal Center B-lymphocytes

    PubMed Central

    van Nierop, Kirsten; Muller, Femke J.M.; Stap, Jan; Van Noorden, Cornelis J.F.; van Eijk, Marco; de Groot, Cornelis

    2006-01-01

    During germinal center (GC) reactions, B-lymphocytes with high-affinity B-cell receptors are selected. Regulation of apoptosis is a key process in selecting such wanted B-cells and in eliminating B-cells with unwanted specificities. In this paper, we show that apoptosis in human GC B-cells involves lysosomal destabilization, which is strictly controlled by caspase-8 activity, but not by caspase-9 activity. Ligation of CD40 provides resistance to lysosomal destabilization. Experimental lysosomal rupture by the lysosomotropic drug O-methyl-L-serine dodecylamide hydrochloride (MSDH) induces apoptosis in GC B-cells, including phosphatidyl serine exposure, mitochondrial inactivation, and DNA fragmentation. These apoptotic features occur in the absence of caspase-3 activity. Follicular dendritic cells (FDCs) protect binding B-lymphocytes from lysosomal destabilization, in both the absence and the presence of MSDH. Our study demonstrates that lysosomal leakage induces apoptosis of GC B-cells in a caspase-3-independent manner and that high-affinity binding to FDCs prevents lysosomal leakage and apoptosis in GC B-cells. PMID:16957167

  16. Siva is an apoptosis-selective p53 target gene important for neuronal cell death

    Microsoft Academic Search

    S B R Jacobs; S Basak; J I Murray; N Pathak; L D Attardi

    2007-01-01

    p53 plays a central role in neuronal cell death resulting from acute injury or disease. To define the pathway by which p53 triggers apoptosis, we used microarray analysis to identify p53 target genes specifically upregulated during apoptosis but not cell cycle arrest. This analysis identified a small subset of targets highly selective for the p53 apoptotic response, including Siva, a

  17. Profit-Optimal Model and Target Size Selection with Variable Marginal Costs

    Microsoft Academic Search

    Alan Abrahams; Firass Hathout; Andreas Staubli; Balaji Padmanabhan

    Various organizations, from catalogue order companies to credit card and insurance institutions, employ direct-mail -response as a core marketing strategy. As the demand of a given random selection of prospects is uncertain, many of these corporations use data mining techniques to characterize good prospects in their target audiences and improve the likelihood of response. Conventional approaches to model and target

  18. Neuronal activity representing visuospatial mnemonic processes associated with target selection in the monkey dorsolateral prefrontal cortex

    Microsoft Academic Search

    Michiyo Iba; Toshiyuki Sawaguchi

    2002-01-01

    To investigate how visuospatial mnemonic and target selection processes are represented in the dorsolateral prefrontal cortex (PFC), we studied neuronal attributes of the dorsolateral PFC while monkeys were performing oculomotor delayed visual search (ODVS) and oculomotor delayed-response (ODR) tasks. In the ODVS task, the subject made a memory-guided saccade to a remembered target location that had been presented along with

  19. Tumor-Selective Radiopharmaceutical Targeting via Receptor-Mediated Endocytosis of Gallium67-Deferoxamine-Folate

    Microsoft Academic Search

    Carla J. Mathias; Susan Wang; Robert J. Lee; David J. Waters; Philip S. Low; Mark A. Green

    The receptor-mediated endocytosis uptake pathway for the vitamin folate was investigated as a target for tumor-selective radiopharma- ceutical delivery. The molecular target for this delivery mechanism is a membrane-associated folate binding protein (FBP)that is overex- pressed by a variety of malignant cell lines. Methods: The ability of a 67Ga-labeled deferoxamine-folate conjugate (67Ga-DF-folate)to target tumor cells in vivo was examined using

  20. 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

  1. Cell-SELEX-based selection of aptamers that recognize distinct targets on metastatic colorectal cancer cells.

    PubMed

    Li, Wan-Ming; Bing, Tao; Wei, Jia-Yi; Chen, Zhe-Zhou; Shangguan, Di-Hua; Fang, Jin

    2014-08-01

    The development of diagnostic/therapeutic strategies against metastasis-related molecular targets is critical for improving the survival rate of cancer patients. Subtractive Cell-SELEX was performed using highly metastatic colorectal cancer (CRC) LoVo cells and non-metastatic HCT-8 cells as the target and negative cells, respectively, for the selection of metastatic-specific aptamers. This process generated seven aptamers that displayed highly specific binding to the target cells with Kds in the nanomolar range. Based on the distinct chemical/biological properties of their individual cell surface targets, the aptamers were separately functionalized: the receptor-targeting aptamer W14 was used as a carrier for doxorubicin, resulting in the specific delivery of the drug to the target cells and a significant reduction of its cytotoxicity to non-target cells, and the non-receptor-binding aptamer W3 was used as a molecular probe conjugated to quantum dots for the targeted imaging of metastatic cancer cell lines, spontaneous lung metastasis murine tissue, and metastatic CRC patient tissues. In addition, these aptamers can be used in combination due to their lack of detectable mutual-binding interference. The study demonstrates that a panel of aptamers that recognize distinct features of target molecules can be obtained through single Cell-SELEX selection, and the selected aptamers may be individually functionalized for specific applications and/or utilized in combination. PMID:24857291

  2. Targets of recent positive selection in Indian populations

    E-print Network

    Romero, Irene Gallego

    2010-06-24

    chosen ‘core’ SNP Other related metrics: iHS: integral under the EHH curve, sensitive to allelic ancestry XP-EHH: cross population EHH, compares population pairs, detects the action of selection in one population but not the other Sample composition 156... : Inferring haplotype from genotype Calculating test statistics: iHS and XP-EHH Data post-processing: ~550,000 data points per population per statistic SNPs to genes/genomic regions Phasing Likelihood-based methods 550,000 SNPs per individual, ~1...

  3. Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes

    PubMed Central

    de Groote, Marloes L.; Verschure, Pernette J.; Rots, Marianne G.

    2012-01-01

    Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined DNA sequences are uniquely suited to answer such questions and could provide potent (bio)medical tools. Toward the goal of gene-specific GEM by overwriting epigenetic marks (Epigenetic Editing, EGE), instructive epigenetic marks need to be identified and their writers/erasers should then be fused to gene-specific DNA binding domains. The appropriate epigenetic mark(s) to change in order to efficiently modulate gene expression might have to be validated for any given chromatin context and should be (mitotically) stable. Various insights in such issues have been obtained by sequence-specific targeting of epigenetic enzymes, as is presented in this review. Features of such studies provide critical aspects for further improving EGE. An example of this is the direct effect of the edited mark versus the indirect effect of recruited secondary proteins by targeting epigenetic enzymes (or their domains). Proof-of-concept of expression modulation of an endogenous target gene is emerging from the few EGE studies reported. Apart from its promise in correcting disease-associated epi-mutations, EGE represents a powerful tool to address fundamental epigenetic questions. PMID:23002135

  4. Bowel microorganisms--a target for selective antimicrobial control.

    PubMed

    van Saene, H K; Percival, A

    1991-09-01

    This article reviews the eight factors that determine the outcome of selective antimicrobial control (SAC) a technique aimed at the clearance of intestinal Gram-negative bacillary carriage by means of lethal faecal anti-microbial concentrations. They are as follows: (i) the carrier state; (ii) compliance; (iii) SAC aiming at prophylaxis vs treatment; (iv) minimum bactericidal concentration (MBC) of the antimicrobial; (v) dosage; (vi) pharmacokinetics; (vii) faecal inactivation; and (viii) microorganisms to be controlled. In the second part, non-absorbable SAC regimens are compared with absorbable trimethoprim/sulphamethoxazole (TMP/SMZ) and the fluoroquinolones in different clinical settings including neutropenia, intensive care, hepatic encephalopathy, liver transplantation and the salmonella carrier state. Ablation of gut carriage and superinfections are the main endpoints reviewed in this article. The newer fluoroquinolones are potent SAC agents to deal with enterobacteria. Pseudomonads are the major gap in their SAC spectrum. TMP/SMZ emerges as a SAC agent of limited value, whilst the newer non-absorbable combination of polymyxin/tobramycin seems to be the most potent SAC programme since it has activity against pseudomonads. In a third part, three current issues--the emergence of resistance, the selectivity and the tissue effect are discussed. Finally, a potent fluoroquinolone combined with oral polymyxin/tobramycin seems to be the most effective SAC programme currently available to control enterobacteria and pseudomonads in patients in whom bacterial translocation is a risk with minimal risk of resistance emerging. PMID:1684194

  5. Selection of targets and ion sources for RIB generation at the Holifield Radioactive Ion Beam Facility

    SciTech Connect

    Alton, G.D.

    1995-12-31

    In this report, the authors describe the performance characteristics for a selected number of target ion sources that will be employed for initial use at the Holifield Radioactive Ion Beam Facility (HRIBF) as well as prototype ion sources that show promise for future use for RIB applications. A brief review of present efforts to select target materials and to design composite target matrix/heat-sink systems that simultaneously incorporate the short diffusion lengths, high permeabilities, and controllable temperatures required to effect fast and efficient diffusion release of the short-lived species is also given.

  6. 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

  7. Common Risk Alleles for Inflammatory Diseases Are Targets of Recent Positive Selection

    E-print Network

    Raychaudhuri, Soumya

    .16,17 Similarly, it was shown that the positively selected celiac disease (MIM 212750) risk variantARTICLE Common Risk Alleles for Inflammatory Diseases Are Targets of Recent Positive Selection identified hundreds of loci harboring genetic variation influencing inflammatory- disease susceptibility

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

    PubMed Central

    Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C.D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

    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

  9. Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis.

    PubMed

    Polishchuk, Elena V; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C D; Chan, Jefferson; Chang, Christopher J; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S

    2014-06-23

    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

  10. Lysosomes integrate metabolic-inflammatory cross-talk in primary macrophage inflammasome activation.

    PubMed

    Weber, Kassandra; Schilling, Joel D

    2014-03-28

    Macrophage dysfunction and inflammasome activation have been implicated in the pathogenesis of diabetes and its complications. Prolonged inflammation and impaired healing are hallmarks of the diabetic response to tissue injury, and excessive inflammasome activation has been associated in these phenotypes. However, the mechanisms that regulate the inflammasome in response to lipid metabolic and inflammatory stress are incompletely understood. We have shown previously that IL-1? secretion is induced in primary macrophages exposed to the dietary saturated fatty acid palmitate in combination with LPS. In this study, we sought to unravel the mechanisms underlying the activation of this lipotoxic inflammasome. We demonstrate that palmitate-loaded primary macrophages challenged with LPS activate the NLRP3 inflammasome through a mechanism that involves the lysosome. Interestingly, the lysosome was involved in both the regulation of pro-IL-1? levels and its subsequent cleavage/release. The lysosomal protease cathepsin B was required for IL-1? release but not pro-IL-1? production. In contrast, disrupting lysosomal calcium regulation decreased IL-1? release by reducing pro-IL-1? levels. The calcium pathway involved the calcium-activated phosphatase calcineurin, which stabilized IL-1? mRNA. Our findings provide evidence that the lysosome plays a key role in both the priming and assembly phases of the lipostoxic inflammasome. These findings have potential relevance to the hyperinflammatory phenotypes observed in diabetics during tissue damage or infection and identify lysosomes and calcineurin as potential therapeutic targets. PMID:24532802

  11. Lysosomes Integrate Metabolic-Inflammatory Cross-talk in Primary Macrophage Inflammasome Activation*

    PubMed Central

    Weber, Kassandra; Schilling, Joel D.

    2014-01-01

    Macrophage dysfunction and inflammasome activation have been implicated in the pathogenesis of diabetes and its complications. Prolonged inflammation and impaired healing are hallmarks of the diabetic response to tissue injury, and excessive inflammasome activation has been associated in these phenotypes. However, the mechanisms that regulate the inflammasome in response to lipid metabolic and inflammatory stress are incompletely understood. We have shown previously that IL-1? secretion is induced in primary macrophages exposed to the dietary saturated fatty acid palmitate in combination with LPS. In this study, we sought to unravel the mechanisms underlying the activation of this lipotoxic inflammasome. We demonstrate that palmitate-loaded primary macrophages challenged with LPS activate the NLRP3 inflammasome through a mechanism that involves the lysosome. Interestingly, the lysosome was involved in both the regulation of pro-IL-1? levels and its subsequent cleavage/release. The lysosomal protease cathepsin B was required for IL-1? release but not pro-IL-1? production. In contrast, disrupting lysosomal calcium regulation decreased IL-1? release by reducing pro-IL-1? levels. The calcium pathway involved the calcium-activated phosphatase calcineurin, which stabilized IL-1? mRNA. Our findings provide evidence that the lysosome plays a key role in both the priming and assembly phases of the lipostoxic inflammasome. These findings have potential relevance to the hyperinflammatory phenotypes observed in diabetics during tissue damage or infection and identify lysosomes and calcineurin as potential therapeutic targets. PMID:24532802

  12. 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

  13. A non-conserved miRNA regulates lysosomal function and impacts on a human lysosomal storage disorder.

    PubMed

    Frankel, Lisa B; Di Malta, Chiara; Wen, Jiayu; Eskelinen, Eeva-Liisa; Ballabio, Andrea; Lund, Anders H

    2014-01-01

    Sulfatases are key enzymatic regulators of sulfate homeostasis with several biological functions including degradation of glycosaminoglycans (GAGs) and other macromolecules in lysosomes. In a severe lysosomal storage disorder, multiple sulfatase deficiency (MSD), global sulfatase activity is deficient due to mutations in the sulfatase-modifying factor 1 (SUMF1) gene, encoding the essential activator of all sulfatases. We identify a novel regulatory layer of sulfate metabolism mediated by a microRNA. miR-95 depletes SUMF1 protein levels and suppresses sulfatase activity, causing the disruption of proteoglycan catabolism and lysosomal function. This blocks autophagy-mediated degradation, causing cytoplasmic accumulation of autophagosomes and autophagic substrates. By targeting miR-95 in cells from MSD patients, we can effectively increase residual SUMF1 expression, allowing for reactivation of sulfatase activity and increased clearance of sulfated GAGs. The identification of this regulatory mechanism opens the opportunity for a unique therapeutic approach in MSD patients where the need for exogenous enzyme replacement is circumvented. PMID:25524633

  14. Lysosomes and ?-synuclein form a dangerous duet leading to neuronal cell death

    PubMed Central

    Bourdenx, Mathieu; Bezard, Erwan; Dehay, Benjamin

    2014-01-01

    Neurodegenerative diseases are (i) characterized by a selective neuronal vulnerability to degeneration in specific brain regions; and (ii) likely to be caused by disease-specific protein misfolding. Parkinson’s disease (PD) is characterized by the presence of intraneuronal proteinacious cytoplasmic inclusions, called Lewy Bodies (LB). ?-Synuclein, an aggregation prone protein, has been identified as a major protein component of LB and the causative for autosomal dominant PD. Lysosomes are responsible for the clearance of long-lived proteins, such as ?-synuclein, and for the removal of old or damaged organelles, such as mitochondria. Interestingly, PD-linked ?-synuclein mutants and dopamine-modified wild-type ?-synuclein block its own degradation, which result in insufficient clearance, leading to its aggregation and cell toxicity. Moreover, both lysosomes and lysosomal proteases have been found to be involved in the activation of certain cell death pathways. Interestingly, lysosomal alterations are observed in the brains of patients suffering from sporadic PD and also in toxic and genetic rodent models of PD-related neurodegeneration. All these events have unraveled a causal link between lysosomal impairment, ?-synuclein accumulation, and neurotoxicity. In this review, we emphasize the pathophysiological mechanisms connecting ?-synuclein and lysosomal dysfunction in neuronal cell death. PMID:25177278

  15. [Lysosomal storage diseases--an overview].

    PubMed

    Kloska, Anna; Tylki-Szyma?ska, Anna; Wegrzyn, Grzegorz

    2011-01-01

    Lysosomal storage diseases (LSD) are a group of fifty or so metabolic disorders. They are caused by genetic defects causing a lack or severe deficiency in activity of one of proteins belonging to four functional groups: acid lysosomal hydorlases involved in degradation of various macromolecules, proteins involved in lysosomal transportation, proteins required to deliver enzymes into lysosomes or activators of lysosomal enzymes. Due to their deficiency, undegraded or partially degraded macromolecules accumulates in lysosomes, causing dysfunction of cells, tissues and organs. Most LSDs are inherited in autosomal recessive manner. There are only three exceptions: Fabry disease and Hunter disease (mucopolysaccharidosis type II), which are X-linked recessive disorders, and Danon disease, which is an X-linked and dominant. As significant advantages have been achieved in understanding of LSD pathomechanisms, these diseases became examples of the importance of biochemical, genetic, molecular and biotechnological studies in development of diagnostics, understanding of biological mechanisms of diseases and development of modern therapeutical methods. In this article, an overview on LSD is presented, while more detailed description of some groups of these diseases: lipidoses, glycogenoses and mucopolysaccharidoses, will be presented in subsequent articles included in this issue. PMID:21913413

  16. In vitro selection of ssDNA aptamers using biotinylated target proteins.

    PubMed

    Mayer, Günter; Höver, Thomas

    2009-01-01

    Aptamers are single-stranded nucleic acids that bind specifically to a target molecule and thus often inhibit target-associated biological functions. Aptamers have been described for a series of target molecules including peptides, proteins, and even living cells. Besides RNA and 20-modified RNA molecules also ssDNA molecules can be subjected to in vitro selection protocols aiming at the enrichment of ssDNA aptamers. ssDNA aptamers can be selected using the SELEX procedure (systematic enrichment of ligands by exponential amplification) from libraries of randomized single-stranded DNA with a diversity of up to 10(16) different molecules. In repetitive selection cycles, the library is incubated with the target of choice and separation of non-binding sequences from bound sequences is achieved by distinct separation methods. The bound molecules are specifically eluted and amplified, thus representing the starting library for the next cycle. Thereby, an enriched population of aptamers is evolved. Here we describe a generalized in vitro selection experiment aiming at the enrichment of ssDNA aptamers using biotinylated target molecules. This procedure allows the application of streptavidin-biotin chemistry to separate bound from unbound DNA species during the selection process. PMID:19377986

  17. 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.

  18. Phylogeny-driven target selection for large-scale genome-sequencing (and other) projects

    PubMed Central

    Göker, Markus; Klenk, Hans-Peter

    2013-01-01

    Despite the steadily decreasing costs of genome sequencing, prioritizing organisms for sequencing remains important in large-scale projects. Phylogeny-based selection is of interest to identify those organisms whose genomes can be expected to differ most from those that have already been sequenced. Here, we describe a method that infers a phylogenetic scoring independent of which set of organisms has previously been targeted, which is computationally simple and easy to apply in practice. The scoring itself, as well as pre- and post-processing of the data, is illustrated using two real-world examples in which the method has already been applied for selecting targets for genome sequencing. These projects are the JGI CSP Genomic Encyclopedia of Bacteria and Archaea phase I, targeting 1,000 type strains, and, on a smaller-scale, the phylogenomics of the Roseobacter clade. Potential artifacts of the method are discussed and compared to a selection approach based on the taxonomic classification. PMID:23991265

  19. Phylogeny-driven target selection for large-scale genome-sequencing (and other) projects.

    PubMed

    Göker, Markus; Klenk, Hans-Peter

    2013-01-01

    Despite the steadily decreasing costs of genome sequencing, prioritizing organisms for sequencing remains important in large-scale projects. Phylogeny-based selection is of interest to identify those organisms whose genomes can be expected to differ most from those that have already been sequenced. Here, we describe a method that infers a phylogenetic scoring independent of which set of organisms has previously been targeted, which is computationally simple and easy to apply in practice. The scoring itself, as well as pre- and post-processing of the data, is illustrated using two real-world examples in which the method has already been applied for selecting targets for genome sequencing. These projects are the JGI CSP Genomic Encyclopedia of Bacteria and Archaea phase I, targeting 1,000 type strains, and, on a smaller-scale, the phylogenomics of the Roseobacter clade. Potential artifacts of the method are discussed and compared to a selection approach based on the taxonomic classification. PMID:23991265

  20. The effect of mean luminance on the size selectivity of identified target interneurons in the dragonfly

    Microsoft Academic Search

    Robert M. Olberg; Robert B. Pinter

    1990-01-01

    1.By penetrating axons in the ventral nerve cord of the dragonfly, Aeshna umbrosa, we measured the intracellular responses of target-selective visual interneurons to movement of black square ‘targets’ ranging from 1° to 32° visual angle at several levels of mean background luminance.2.Neuronal responses, measured both in number of spikes and in the magnitude of integrated postsynaptic potentials, showed a preference

  1. 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

  2. Comparison of the cancer gene targeting and biochemical selectivities of all targeted kinase inhibitors approved for clinical use.

    PubMed

    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

  3. Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

    PubMed Central

    Mukherjee, Amarnath; Narayan, Kumar P; Pal, Krishnendu; Kumar, Jerald M; Rangaraj, Nandini; Kalivendi, Shasi V; Banerjee, Rajkumar

    2009-01-01

    Glucocorticoid receptors (GRs) are ubiquitous, nuclear hormone receptors residing in cell types of both cancer and noncancerous origin. It is not known whether cancer cell–associated GR alone can be selectively manipulated for delivery of exogenous genes to its nucleus for eliciting anticancer effect. We find that GR ligand, dexamethasone (Dex) in association with cationic lipoplex (termed as targeted lipoplex) could selectively manipulate GR in cancer cells alone for the delivery of transgenes in the nucleus, a phenomenon that remained unobserved in normal cells. The targeted lipoplex (i) showed GR-targeted transfections in all cancer cells experimented (P < 0.01), (ii) significantly diminished transfection in cancer cells when GR is downregulated (P < 0.01), and (iii) elicited specific nuclear translocation of targeted lipoplex in cancer cells, followed by upregulated transactivation of glucocorticoid response element (GRE)– promoted gene. Using anticancer gene, targeted lipoplex induced significant tumor growth retardation in mice in comparison to different control groups (P < 0.05). Interestingly, cell surface–associated Hsp90 in cancer cells assisted the intracellular uptake of GR-targeted lipoplex. Moreover, selective inhibition of Hsp90 in noncancer cells resulted in cancer cell-like, aberrant, GR activation. The current study discovers a therapeutically important, unique property of cancer cell associated–GR that may be linked to a compromised role of Hsp90. PMID:19223869

  4. Rapid parallel attentional target selection in single-color and multiple-color visual search.

    PubMed

    Grubert, Anna; Eimer, Martin

    2015-02-01

    Previous work has demonstrated that when targets are defined by a constant feature, attention can be directed rapidly and in parallel to sequentially presented target objects at different locations. We assessed how fast attention is allocated to multiple objects when this process cannot be controlled by a unique color-specific attentional template. N2pc components were measured as temporal markers of the attentional selection of 2 color-defined targets that were presented in rapid succession. Both targets either had the same color (one color task) or differed in color (two color task). Although there were small but systematic delays of target selection in the two color task relative to the one color task, attention was allocated extremely rapidly to both target objects in the two color task, which is inconsistent with the hypothesis that their selection was based on a slow switch between different color templates. Two follow-up experiments demonstrated that these delays did not reflect template switch costs, but were the result of competitive interactions between simultaneously active attentional templates. These results show that the control of focal attention during multiple-feature search operates much faster and more flexibly than is usually assumed. PMID:25485665

  5. Software for selecting the most informative sets of genomic loci for multi-target microbial typing

    PubMed Central

    2013-01-01

    Background High-throughput sequencing can identify numerous potential genomic targets for microbial strain typing, but identification of the most informative combinations requires the use of computational screening tools. This paper describes novel software – Automated Selection of Typing Target Subsets (AuSeTTS) - that allows intelligent selection of optimal targets for pathogen strain typing. The objective of this software is to maximise both discriminatory power, using Simpson’s index of diversity (D), and concordance with existing typing methods, using the adjusted Wallace coefficient (AW). The program interrogates molecular typing results for panels of isolates, based on large target sets, and iteratively examines each target, one-by-one, to determine the most informative subset. Results AuSeTTS was evaluated using three target sets: 51 binary targets (13 toxin genes, 16 phage-related loci and 22 SCCmec elements), used for multilocus typing of 153 methicillin-resistant Staphylococcus aureus (MRSA) isolates; 17 MLVA loci in 502 Streptococcus pneumoniae isolates from the MLVA database (http://www.mlva.eu) and 12 MLST loci for 98 Cryptococcus spp. isolates. The maximum D for MRSA, 0.984, was achieved with a subset of 20 targets and a D value of 0.954 with 7 targets. Twelve targets predicted MLST with a maximum AW of 0.9994. All 17 S. pneumoniae MLVA targets were required to achieve maximum D of 0.997, but 4 targets reached D of 0.990. Twelve targets predicted pneumococcal serotype with a maximum AW of 0.899 and 9 predicted MLST with maximum AW of 0.963. Eight of the 12 MLST loci were sufficient to achieve the maximum D of 0.963 for Cryptococcus spp. Conclusions Computerised analysis with AuSeTTS allows rapid selection of the most discriminatory targets for incorporation into typing schemes. Output of the program is presented in both tabular and graphical formats and the software is available for free download from http://www.cidmpublichealth.org/pages/ausetts.html. PMID:23635100

  6. Topical liposome targeting of dyes, melanins, genes, and proteins selectively to hair follicles.

    PubMed

    Hoffman, R M

    1998-01-01

    For therapeutic and cosmetic modification of hair, we have developed a hair-follicle-selective macromolecule and small molecule targeting system with topical application of phosphatidylcholine-based liposomes. Liposome-entrapped melanins, proteins, genes, and small-molecules have been selectively targeted to the hair follicle and hair shafts of mice. Liposomal delivery of these molecules is time dependent. Negligible amounts of delivered molecules enter the dermis, epidermis, or bloodstream thereby demonstrating selective follicle delivery. Naked molecules are trapped in the stratum corneum and are unable to enter the follicle. The potential of the hair-follicle liposome delivery system for therapeutic use for hair disease as well as for cosmesis has been demonstrated in 3-dimensional histoculture of hair-growing skin and mouse in vivo models. Topical liposome selective delivery to hair follicles has demonstrated the ability to color hair with melanin, the delivery of the active lac-Z gene to hair matrix cells and delivery of proteins as well. Liposome-targeting of molecules to hair follicles has also been achieved in human scalp in histoculture. Liposomes thus have high potential in selective hair follicle targeting of large and small molecules, including genes, opening the field of gene therapy and other molecular therapy of the hair process to restore hair growth, physiologically restore or alter hair pigment, and to prevent or accelerate hair loss. PMID:9588863

  7. Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance

    PubMed Central

    Medina, Diego L.; Fraldi, Alessandro; Bouche, Valentina; Annunziata, Fabio; Mansueto, Gelsomina; Spampanato, Carmine; Puri, Claudia; Pignata, Antonella; Martina, Jose A.; Sardiello, Marco; Palmieri, Michela; Polishchuk, Roman; Puertollano, Rosa; Ballabio, Andrea

    2011-01-01

    Summary Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca2+ levels through the activation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular storage. PMID:21889421

  8. Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes

    Microsoft Academic Search

    Carsten Hopf; Mikhail M Savitski; Antje Dittmann; Paola Grandi; Anne-Marie Michon; Judith Schlegl; Yann Abraham; Isabelle Becher; Giovanna Bergamini; Markus Boesche; Manja Delling; Birgit Dümpelfeld; Dirk Eberhard; Carola Huthmacher; Toby Mathieson; Daniel Poeckel; Valérie Reader; Katja Strunk; Gavain Sweetman; Ulrich Kruse; Gitte Neubauer; Nigel G Ramsden; Marcus Bantscheff; Gerard Drewes

    2011-01-01

    The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including

  9. Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay

    Microsoft Academic Search

    Katherine Watt Chan; James Waire; Betsy Simons; Ken Karey; John Fung; Diane Copeland; Laura Andrews

    2004-01-01

    Lysosomal acid ?-glucocerebrosidase hydrolyzes glucocerebroside to glucose ceramide. Patients diagnosed with Gaucher disease, however, lack this enzyme, leading to the accumulation of glucocerebroside in tissue macrophages within multiple organs. Such patients can receive enzyme replacement therapy during which a human placental-derived or recombinant form of acid ?-glucocerebrosidase is targeted to the macrophages. As part of evaluating the effectiveness of such

  10. Inhibition of autophagic-lysosomal delivery and autophagic lactolysis by asparagine

    PubMed Central

    1991-01-01

    Overall autophagy was measured in isolated hepatocytes as the sequestration and lysosomal hydrolysis of electroinjected [14C]lactose, using HPLC to separate the degradation product [14C]glucose from undegraded lactose. In addition, the sequestration step was measured separately as the transfer from cytosol to sedimentable cell structures of electroinjected [3H]raffinose or endogenous lactate dehydrogenase (LDH; in the presence of leupeptin to inhibit lysosomal proteolysis). Inhibitor effects at postsequestrational steps could be detected as the accumulation of autophaged lactose (which otherwise is degraded intralysosomally), or of LDH in the absence of leupeptin. Asparagine, previously shown to inhibit autophagic but not endocytic protein breakdown, strongly suppressed the autophagic hydrolysis of electroinjected lactose. Vinblastine, which inhibits both types of degradation, likewise suppressed lactose hydrolysis. Asparagine had little or no effect on sequestration, but caused an accumulation of autophaged LDH and lactose, indicating inhibition at a postsequestrational step. Neither asparagine nor vinblastine affected the degradation of intralysosomal lactose preaccumulated in the presence of the reversible lysosome inhibitor propylamine. However, if lactose was preaccumulated in the presence of asparagine, both asparagine and vinblastine suppressed its subsequent degradation. The data thus indicate that autophagic-lysosomal delivery, i.e., the transfer of autophaged material from prelysosomal vacuoles to lysosomes, is inhibited selectively by asparagine and non-selectively by vinblastine. PMID:1904444

  11. Abnormal autophagy, ubiquitination, inflammation and apoptosis are dependent upon lysosomal storage and are useful biomarkers of mucopolysaccharidosis VI

    PubMed Central

    Tessitore, Alessandra; Pirozzi, Marinella; Auricchio, Alberto

    2009-01-01

    Background Lysosomal storage diseases are characterized by intracellular accumulation of metabolites within lysosomes. Recent evidence suggests that lysosomal storage impairs autophagy resulting in accumulation of polyubiquitinated proteins and dysfunctional mitochondria, ultimately leading to apoptosis. We studied the relationship between lysosome storage and impairment of different intracellular pathways and organelle function in mucopolysaccharidosis VI, which is characterized by accumulation of dermatan sulfate and signs of visceral and skeletal but not cerebral involvement. Results We show lysosomal storage, impaired autophagy, accumulation of polyubiquitinated proteins, and mitochondrial dysfunction in fibroblasts from mucopolysaccharidosis VI patients. We observe similar anomalies, along with inflammation and cell death, in association with dermatan sulfate storage in the visceral organs of mucopolysaccharidosis VI rats, but not in their central nervous system where dermatan sulfate storage is absent. Importantly, we show that prevention of dermatan sulfate storage in the mucopolysaccharidosis VI rat visceral organs by gene transfer results in correction of abnormal autophagy, inflammation, and apoptosis, suggesting that dermatan sulfate accumulation impairs lysosomal ability to receive and degrade molecules and organelles from the autophagic pathway, thus leading to cell toxicity. Conclusion These results indicate that the non-lysosomal degradation pathways we found activated in mucopolysaccharidosis VI can be both targets of new experimental therapies and biomarkers for follow-up of existing treatments. PMID:19531206

  12. Frontoparietal theta activity supports behavioral decisions in movement-target selection

    PubMed Central

    Rawle, Christian J.; Miall, R. Chris; Praamstra, Peter

    2012-01-01

    There is recent EEG evidence describing task-related changes of theta power in spatial attention and reaching/pointing tasks. Here, we aim to better characterize this theta activity and determine whether it is associated with visuospatial memory or with visuospatial selection functions of the frontoparietal cortex. We recorded EEG from 20 participants during a movement precuing task with center-out joystick movements. Precues displayed 1, 2, or 4 potential targets and were followed (stimulus onset asynchrony 1.2 s) by a central response cue indicating the movement-target. Remembering the precued target location(s) was mandatory in one and optional in a second version of the task. Analyses evaluated two slow brain potentials (CNV, contingent negative variation and CDA, contralateral delay activity) and task-related power changes. Results showed a differential modulation of frontal CNV and parietal CDA, consistent with earlier described set-size effects on motor preparation and visual short-term memory. Short-lived phases of theta event-related synchronization (ERS) were found 150–500 ms after precue and response cue presentation, exhibiting parietal and frontal maxima. The increase of frontoparietal theta power following response cue presentation was strongly modulated by target load, i.e., absent for 1-target (when the movement-target could be selected in advance), contrasting with a robust 20–50% ERS response in 2- and 4-target conditions. The scalp distribution, the timing, and the modulation by set-size suggest a role of theta activity in movement-target selection. The results support a recently proposed view of theta as emerging around behavioral decision points, linked to the evaluation of choice-relevant information. PMID:22629241

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

    PubMed

    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

  14. Promoter-targeted Phage Display Selections with Preassembled Synthetic Zinc Finger Libraries for Endogenous Gene Regulation

    Microsoft Academic Search

    Caren V. Lund; Pilar Blancafort; Mikhail Popkov; Carlos F. Barbas III

    2004-01-01

    Regulation of endogenous gene expression has been achieved using synthetic zinc finger proteins fused to activation or repression domains, zinc finger transcription factors (TFZFs). Two key aspects of selective gene regulation using TFZFs are the accessibility of a zinc finger protein to its target DNA sequence and the interaction of the fused activation or repression domain with endogenous proteins. Previous

  15. aB-Crystallin Selectively Targets Intermediate Filament Proteins during Thermal Stress

    E-print Network

    Clark, John

    B-crystallin in lens cell homogenates during conditions of mild thermal stress. METHODS. The authors report the useaB-Crystallin Selectively Targets Intermediate Filament Proteins during Thermal Stress PaulHsp) expressed at high levels in the lens of the eye, where its molecular chaperone functions may protect against

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

    SciTech Connect

    Zasowski, G.; Johnson, Jennifer A.; Andrews, B.; Epstein, C. [Department of Astronomy, Ohio State University, Columbus, OH 43210 (United States); Frinchaboy, P. M.; Jackson, K. [Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX 76129 (United States); Majewski, S. R.; Chojnowski, S. D.; Skrutskie, M. F.; Beaton, R. L. [Department of Astronomy, University of Virginia, Charlottesville, VA 22904 (United States); Nidever, D. L. [Department of Astronomy, University of Michigan, Ann Arbor, MI 48109 (United States); Pinto, H. J. Rocha; Girardi, L. [Laboratorio Interinstitucional de e-Astronomia-LIneA, Rio de Janeiro, RJ 20921-400 (Brazil); Cudworth, K. M. [Yerkes Observatory, University of Chicago, Williams Bay, WI 53191 (United States); Munn, J. [US Naval Observatory, Flagstaff Station, Flagstaff, AZ 86001 (United States); Blake, C. H. [Department of Astrophysical Sciences, Princeton University, Princeton, NJ 08544 (United States); Covey, K. [Lowell Observatory, Flagstaff, AZ 86001 (United States); Deshpande, R.; Fleming, S. W. [Department of Astronomy and Astrophysics, Pennsylvania State University, University Park, PA 16802 (United States); Fabbian, D., E-mail: gail.zasowski@gmail.com [Instituto de Astrofisica de Canarias, Calle Via Lactea s/n, E-38205 La Laguna, Tenerife (Spain); 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.

  17. Distance factors and target market selection: the moderating effect of market potential

    Microsoft Academic Search

    Shavin Malhotra; K. Sivakumar; PengCheng Zhu

    2009-01-01

    Purpose – The paper is in the domain of marketing strategies of multinational firms. Specifically, it aims to focus on target market selection of multinational firms. Design\\/methodology\\/approach – Using the cultural, administrative, geographic, and economic distance framework proposed by Ghemawat, the authors offer empirical support for the role of different distance factors on firms' foreign market acquisition behavior. In addition,

  18. Audience Selection for On-line Brand Advertising: Privacy-friendly Social Network Targeting

    E-print Network

    Provost, Foster

    Audience Selection for On-line Brand Advertising: Privacy-friendly Social Network Targeting Foster advertising. Unlike direct-marketing-style on- line advertising, the goal of on-line brand advertising on user-generated content sites, for the purpose of finding good audiences for brand advertising (as

  19. 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…

  20. Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth

    E-print Network

    Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor, particularly in combination with chemotherapy. Here, we show that low doses of metformin, a standard drug different types of breast cancer. The combination of metformin and a well-defined chemotherapeutic agent

  1. Web-Scale Multi-Task Feature Selection for Behavioral Targeting

    E-print Network

    Tomkins, Andrew

    Web-Scale Multi-Task Feature Selection for Behavioral Targeting Amr Ahmed2 , Mohamed Aly1 optimizing purchase activities, called conversions, faces two main challenges: the web-scale amounts of user and a distributed subgradient oracle. To efficiently handle web-scale user histories, we present a dis- tributed

  2. VizieR Online Data Catalog: SUNS and DEBRIS surveys target selection (Phillips+, 2010)

    NASA Astrophysics Data System (ADS)

    Phillips, N. M.; Greaves, J. S.; Dent, W. R. F.; Matthews, B. C.; Holland, W. S.; Wyatt, M. C.; Sibthorpe, B.

    2012-01-01

    Debris discs - analogous to the asteroid and Kuiper-Edgeworth belts in the Solar system - have so far mostly been identified and studied in thermal emission shortward of 100um. The Herschel space observatory and the Submillimetre Common-User Bolometer Array-2 (SCUBA-2) camera on the James Clerk Maxwell Telescope will allow efficient photometric surveying at 70 to 850um, which allows for the detection of cooler discs not yet discovered, and the measurement of disc masses and temperatures when combined with shorter wavelength photometry. The SCUBA-2 Unbiased Nearby Stars survey (SUNS) and the Disc Emission via a Bias-free Reconnaissance in the Infrared/Submillimetre (DEBRIS) Herschel Open Time Key Project are complementary legacy surveys observing samples of ~500 nearby stellar systems. To maximize the legacy value of these surveys, great care has gone into the target selection process. This paper describes the target selection process and presents the target lists of these two surveys. (7 data files).

  3. In vivo photoacoustic imaging with multiple selective targeting using bioconjugated gold nanorods

    NASA Astrophysics Data System (ADS)

    Wei, Chen-Wei; Liao, Chao-Kang; Chen, Ying-Yi; Wang, Churng-Ren Chris; Ding, Ann-Ann; Shiehd, Dar-Bin; Li, Pai-Chi

    2008-02-01

    In this study, photoacoustic imaging is utilized to probe information from oncogene surface molecules of cancer cell with the aid of specific targeting. The ultimate goal is to provide prediction of clinical outcome and treatment response of anti-cancer drugs. Different from single targeting in most research, we accomplished multiple targeting to obtain a molecular profile potentially representing tumor characteristics or to locate the heterogeneous population in one lesion. By conjugating different antibodies to gold nanorods corresponding to different peak absorption bands, multiple targeting and simultaneous detection with photoacoustic imaging can be achieved with laser irradiation at the respective peak optical absorption wavelength. Her2 and EGFR were chosen as our primary target molecules. The targeting complex was evaluated in two types of oral cancer cells, OECM1 and Cal27. The OECM1 cell line overexpresses Her2 but has low expression of EGFR, while Cal27 cell line expresses both antibodies. Also, the targeting efficacy to OECM1 can be further improved by using mixed nanoprobes. The cancer cells were induced on the back of the mice by subcutaneous injection. The captured images show that both cancer cells exhibit a higher photoacoustic response (maximum 3 dB) than control groups with specific targeting, thus demonstrating the feasibility of multiple selective targeting with bioconjugated gold nanorods. Images of multiple targeting with mixed nanoprobes of OECM1 cells also reveal further enhancement of targeting (4 dB). The results showed potential of in vivo photoacoustic molecular imaging, providing a better guidance for diagnosis and treatment of cancer.

  4. 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

  5. Urinary proteins induce lysosomal membrane permeabilization and lysosomal dysfunction in renal tubular epithelial cells.

    PubMed

    Liu, Wei Jing; Xu, Bi-Hua; Ye, Lin; Liang, Dong; Wu, Hong-Luan; Zheng, Yuan-Yuan; Deng, Jian Kun; Li, Benyi; Liu, Hua-Feng

    2015-03-15

    Lysosomal membrane permeabilization (LMP) has been shown to cause the release of cathepsins and other hydrolases from the lysosomal lumen to the cytosol and initiate a cell death pathway. Whether proteinuria triggers LMP in renal tubular epithelial cells (TECs) to accelerate the progression of renal tubulointerstitial injury remains unclear. In the present study, we evaluated TEC injury as well as changes in lysosomal number, volume, activity, and membrane integrity after urinary protein overload in vivo and in vitro. Our results revealed that neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels were significantly increased in the urine of patients with minimal change nephrotic syndrome (MCNS) and the culture supernatant of HK-2 cells treated by urinary proteins extracted from MCNS patients. Urinary protein overload also induced apoptotic cell death in HK-2 cells. Importantly, we found that lysosomal volume and number were markedly increased in TECs of patients with MCNS and HK-2 cells overloaded with urinary proteins. However, lysosome function, as assessed by proteolytic degradation of DQ-ovalbumin and cathepsin-B and cathepsin-L activities, was decreased in HK-2 cells overloaded with urinary proteins. Furthermore, urinary protein overload led to a diffuse cytoplasmic immunostaining pattern of cathepsin-B and irregular immunostaining of lysosome-associated membrane protein-1, accompanying a reduction in intracellular acidic components, which could be improved by pretreatment with antioxidant. Taken together, our results indicate that overloading of urinary proteins caused LMP and lysosomal dysfunction at least partly via oxidative stress in TECs. PMID:25587119

  6. Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases.

    PubMed

    Lloyd-Evans, Emyr; Platt, Frances M

    2011-08-01

    Disrupted cellular Ca(2+) signaling is believed to play a role in a number of human diseases including lysosomal storage diseases (LSD). LSDs are a group of ?50 diseases caused predominantly by mutations in lysosomal proteins that result in accumulation of macromolecules within the lysosome. We recently reported that Niemann-Pick type C (NPC) is the first human disease to be associated with defective lysosomal Ca(2+) uptake and defective NAADP-mediated lysosomal Ca(2+) release. These defects in NPC cells leads to the disruption in endocytosis and subsequent lipid storage that is a feature of this disease. In contrast, Chediak-Higashi Syndrome cells have been reported to have enhanced lysosomal Ca(2+) uptake whilst the TRPML1 protein defective in mucolipidosis type IV is believed to function as a Ca(2+) channel. In this review we provide a summary of the current knowledge on the role of lysosomal Ca(2+) signaling in the pathogenesis of this group of diseases. PMID:21724254

  7. 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

  8. Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11

    PubMed Central

    Renvoisé, Benoît; Chang, Jaerak; Singh, Rajat; Yonekawa, Sayuri; FitzGibbon, Edmond J; Mankodi, Ami; Vanderver, Adeline; Schindler, Alice B; Toro, Camilo; Gahl, William A; Mahuran, Don J; Blackstone, Craig; Pierson, Tyler Mark

    2014-01-01

    Objective Hereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism. Methods We describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes. Results Fibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ZFYVE26 and the SPG11 protein spatacsin were interdependent. Interpretation Emerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work with Zfyve26?/? mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction. PMID:24999486

  9. Alterations in lysosomal and proteasomal markers in Parkinson's disease: relationship to alpha-synuclein inclusions.

    PubMed

    Chu, Yaping; Dodiya, Hemraj; Aebischer, Patrick; Olanow, C Warren; Kordower, Jeffrey H

    2009-09-01

    We explored the relationship between ubiquitin proteasome system (UPS) and lysosomal markers and the formation of alpha-synuclein (alpha-syn) inclusions in nigral neurons in Parkinson disease (PD). Lysosome Associated Membrane Protein 1(LAMP1), Cathepsin D (CatD), and Heat Shock Protein73 (HSP73) immunoreactivity were significantly decreased within PD nigral neurons when compared to age-matched controls. This decrease was significantly greater in nigral neurons that contained alpha-syn inclusions. Immunoreactivity for 20S proteasome was similarly reduced in PD nigral neurons, but only in cells that contained inclusions. In aged control brains, there is staining for alpha-syn protein, but it is non-aggregated and there is no difference in LAMP1, CatD, HSP73 or 20S proteasome immunoreactivity between alpha-syn positive or negative neuromelanin-laden nigral neurons. Targeting over-expression of mutant human alpha-syn in the rat substantia nigra using viral vectors revealed that lysosomal and proteasomal markers were significantly decreased in the neurons that displayed alpha-syn-ir inclusions. These findings suggest that alpha-syn aggregation is a key feature associated with decline of proteasome and lysosome and support the hypothesis that cell degeneration in PD involves proteosomal and lysosomal dysfunction, impaired protein clearance, and protein accumulation and aggregation leading to cell death. PMID:19505575

  10. Time-dependent effects of hydrophobic amine-containing drugs on lysosome structure and biogenesis in cultured human fibroblasts.

    PubMed

    Logan, Randall; Kong, Alex C; Krise, Jeffrey P

    2014-10-01

    Many weakly basic amine-containing drugs are known to be extensively sequestered in acidic lysosomes by an ion trapping-type mechanism. The entrapment of drugs in lysosomes has been shown to influence drug activity, cancer cell selectivity, and pharmacokinetics and can cause the hyperaccumulation of various lipids associated with lysosomes. In this work, we have investigated the prolonged time-dependent effects of drugs on lysosomal properties. We have evaluated two amine-containing drugs with intermediate (propranolol) and high (halofantrine) relative degrees of lipophilicity. Interestingly, the cellular accumulation kinetics of these drugs exhibited a biphasic characteristic at therapeutically relevant exposure levels with an initial apparent steady-state occurring at 2 days followed by a second stage of enhanced accumulation. We provide evidence that this secondary drug accumulation coincides with the nuclear localization of transcription factor EB, a master regulator of lysosome biogenesis, and the appearance of an increased number of smaller and lipid-laden lysosomes. Collectively, these results show that hydrophobic lysosomotropic drugs can induce their own cellular accumulation in a time-dependent fashion and that this is associated with an expanded lysosomal volume. These results have important therapeutic implications and may help to explain sources of variability in drug pharmacokinetic distribution and elimination properties observed in vivo. PMID:25042198

  11. Peptide nucleic acids targeting ?-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells

    PubMed Central

    MONTAGNER, GIULIA; GEMMO, CHIARA; FABBRI, ENRICA; MANICARDI, ALEX; ACCARDO, IGEA; BIANCHI, NICOLETTA; FINOTTI, ALESSIA; BREVEGLIERI, GIULIA; SALVATORI, FRANCESCA; BORGATTI, MONICA; LAMPRONTI, ILARIA; BRESCIANI, ALBERTO; ALTAMURA, SERGIO; CORRADINI, ROBERTO; GAMBARI, ROBERTO

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine ?-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  12. 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

  13. Comparison of five peptide vectors for improved brain delivery of the lysosomal enzyme arylsulfatase A.

    PubMed

    Böckenhoff, Annika; Cramer, Sandra; Wölte, Philipp; Knieling, Simeon; Wohlenberg, Claudia; Gieselmann, Volkmar; Galla, Hans-Joachim; Matzner, Ulrich

    2014-02-26

    Enzyme replacement therapy (ERT) is a treatment option for lysosomal storage disorders (LSDs) caused by deficiencies of soluble lysosomal enzymes. ERT depends on receptor-mediated transport of intravenously injected recombinant enzyme to lysosomes of patient cells. The blood-brain barrier (BBB) prevents efficient transfer of therapeutic polypeptides from the blood to the brain parenchyma and thus hinders effective treatment of LSDs with CNS involvement. We compared the potential of five brain-targeting peptides to promote brain delivery of the lysosomal enzyme arylsulfatase A (ASA). Fusion proteins between ASA and the protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep peptide (Ang-2), and the receptor-binding domains of human apolipoprotein B (ApoB) and ApoE (two versions, ApoE-I and ApoE-II) were generated. All ASA fusion proteins were enzymatically active and targeted to lysosomes when added to cultured cells. In contrast to wild-type ASA, which is taken up by mannose-6-phosphate receptors, all chimeric proteins were additionally endocytosed via mannose-6-phosphate-independent routes. For ASA-Ang-2, ASA-ApoE-I, and ASA-ApoE-II, uptake was partially due to the low-density lipoprotein receptor-related protein 1. Transendothelial transfer in a BBB cell culture model was elevated for ASA-ApoB, ASA-ApoE-I, and ASA-ApoE-II. Brain delivery was, however, increased only for ASA-ApoE-II. ApoE-II was also superior to wild-type ASA in reducing lysosomal storage in the CNS of ASA-knock-out mice treated by ERT. Therefore, the ApoE-derived peptide appears useful to treat metachromatic leukodystrophy and possibly other neurological disorders more efficiently. PMID:24573272

  14. Selection Strategy to Generate Aptamer Pairs that Bind to Distinct Sites on Protein Targets

    PubMed Central

    Gong, Qiang; Wang, Jinpeng; Ahmad, Kareem M.; Csordas, Andrew; Zhou, Jiehua; Nie, Jeff; Stewart, Ron; Thomson, James A.; Rossi, John J.; Soh, H. Tom

    2012-01-01

    Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present Multivalent Aptamer Isolation SELEX (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2?-fluoro-modified RNA aptamers that specifically recognize the ?V or ?3 subunits of integrin ?V?3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologs. Moreover, we show that these aptamer pairs do not interfere with each other’s binding, and effectively detect the target even in complex mixtures such as undiluted serum. PMID:22624874

  15. 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

    2015-02-01

    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

  16. Recruitment of folliculin to lysosomes supports the amino acid–dependent activation of Rag GTPases

    PubMed Central

    Petit, Constance S.; Roczniak-Ferguson, Agnes

    2013-01-01

    Birt-Hogg-Dubé syndrome, a human disease characterized by fibrofolliculomas (hair follicle tumors) as well as a strong predisposition toward the development of pneumothorax, pulmonary cysts, and renal carcinoma, arises from loss-of-function mutations in the folliculin (FLCN) gene. In this study, we show that FLCN regulates lysosome function by promoting the mTORC1-dependent phosphorylation and cytoplasmic sequestration of transcription factor EB (TFEB). Our results indicate that FLCN is specifically required for the amino acid–stimulated recruitment of mTORC1 to lysosomes by Rag GTPases. We further demonstrated that FLCN itself was selectively recruited to the surface of lysosomes after amino acid depletion and directly bound to RagA via its GTPase domain. FLCN-interacting protein 1 (FNIP1) promotes both the lysosome recruitment and Rag interactions of FLCN. These new findings define the lysosome as a site of action for FLCN and indicate a critical role for FLCN in the amino acid–dependent activation of mTOR via its direct interaction with the RagA/B GTPases. PMID:24081491

  17. 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 ?V-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.

  18. 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 score–adjusted 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

  19. 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 ?-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 ?-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 ?-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

  20. [Fabry disease and cystinosis, two lysosomal diseases: similarities and differences].

    PubMed

    Grünfeld, J-P; Servais, A

    2010-12-01

    Fabry disease and cystinosis are both lysosomal diseases. Some clinical features (such as renal and corneal involvement) are shared by both diseases whereas many other features are different (mode of inheritance, rate of progression, mechanism of lysosomal storage, therapeutic modalities etc.). Intermediary mechanisms that lead from lysosomal overload to lesions and disease are still incompletely understood. PMID:21211669

  1. Pharmacological small molecules for the treatment of lysosomal storage disorders

    Microsoft Academic Search

    B. E. Smid; J. M. F. G. Aerts; R. G. Boot; G. E. Linthorst; C. E. M. Hollak

    2010-01-01

    Importance of the field: Inherited lysosomal storage diseases often cause severe disability and have a devastating effect on quality of life. Enzyme replacement therapy (ERT) forms a cornerstone in the treatment of lysosomal enzyme deficiencies. Although for some lysosomal disorders ERT is lifesaving, important intrinsic restrictions of the approach are limited access of infused enzyme to less accessible body compartments

  2. Gene Transfer Strategies for Correction of Lysosomal Storage Disorders

    Microsoft Academic Search

    Alessandra d’Azzo

    2003-01-01

    Lysosomal storage diseases (LSDs) represent a large group of monogenic disorders of metabolism, which affect approximately 1 in 5,000 live births. LSDs result from a single or multiple deficiency of specific lysosomal hydrolases, the enzymes responsible for the luminal catabolization of macromolecular substrates. The consequent accumulation of undigested metabolites in lysosomes leads to polysystemic dysfunction, including progressive neurologic deterioration, mental

  3. Selective whole genome amplification for resequencing target microbial species from complex natural samples.

    PubMed

    Leichty, Aaron R; Brisson, Dustin

    2014-10-01

    Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory-prepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in >10(5)-fold amplification of the target genomes with <6.7-fold amplification of the background. SWGA-treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs. PMID:25096321

  4. 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.

  5. Cell Reports Presenilin Deficiency or Lysosomal Inhibition

    E-print Network

    De Robertis, Eddy M.

    was found in cells deficient in presenilin, a protein associ- ated with Alzheimer's disease. The Wnt by presenilin deficiency. INTRODUCTION Canonical Wnt signaling is essential for embryonic development, stem cell#12;Cell Reports Article Presenilin Deficiency or Lysosomal Inhibition Enhances Wnt Signaling

  6. Update on treatment of lysosomal storage diseases

    PubMed Central

    Bruni, S; Loschi, L; Incerti, C; Gabrielli, O; Coppa, GV

    2007-01-01

    Summary Lysosomal storage diseases (LSDs) are a large group of disorders caused by a deficiency of specific enzymes responsible for the degradation of substances present in lysosomes. In the past few years, treatments for LSDs were non specific and could only cope with signs and symptoms of the diseases. A successful therapeutic approach to LSDs should instead address to the underlying causes of the diseases, thus helping the degradation of the accumulated metabolites in the various organs, and at the same time preventing their further deposition. One way is to see to an available source of the deficient enzyme: bone marrow transplantation, enzyme replacement therapy and gene therapy are based on this rationale. The purpose of substrate reduction therapy is to down regulate the formation of the lysosomal substance to a rate at which the residual enzyme activity can catabolize the stored and de novo produced lysosomal substrate. Chemical chaperone therapy is based on small molecules able to bind and stabilize the misfolded enzymes. This paper offers a historical overview on the therapeutic strategies for LSDs. PMID:17915580

  7. Structure of a human lysosomal sulfatase

    Microsoft Academic Search

    Charles S Bond; Peter R Clements; Samantha J Ashby; Charles A Collyer; Stephen J Harrop; John J Hopwood; J Mitchell Guss

    1997-01-01

    Background: Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed

  8. 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

  9. Rescue of compromised lysosomes enhances degradation of photoreceptor outer segments and reduces lipofuscin-like autofluorescence in retinal pigmented epithelial cells.

    PubMed

    Guha, Sonia; Liu, Ji; Baltazar, Gabe; Laties, Alan M; Mitchell, Claire H

    2014-01-01

    Healthful cell maintenance requires the efficient degradative processing and removal of waste material. Retinal pigmented epithelial (RPE) cells have the onerous task of degrading both internal cellular debris generated through autophagy as well as phagocytosed photoreceptor outer segments. We propose that the inadequate processing material with the resulting accumulation of cellular waste contributes to the downstream pathologies characterized as age-related macular degeneration (AMD). The lysosomal enzymes responsible for clearance function optimally over a narrow range of acidic pH values; elevation of lysosomal pH by compounds like chloroquine or A2E can impair degradative enzyme activity and lead to a lipofuscin-like autofluorescence. Restoring acidity to the lysosomes of RPE cells can enhance activity of multiple degradative enzymes and is therefore a logical target in early AMD. We have identified several approaches to reacidify lysosomes of compromised RPE cells; stimulation of beta-adrenergic, A2A adenosine and D5 dopamine receptors each lowers lysosomal pH and improves degradation of outer segments. Activation of the CFTR chloride channel also reacidifies lysosomes and increases degradation. These approaches also restore the lysosomal pH of RPE cells from aged ABCA4(-/-) mice with chronically high levels of A2E, suggesting that functional signaling pathways to reacidify lysosomes are retained in aged cells like those in patients with AMD. Acidic nanoparticles transported to RPE lysosomes also lower pH and improve degradation of outer segments. In summary, the ability of diverse approaches to lower lysosomal pH and enhance outer segment degradation support the proposal that lysosomal acidification can prevent the accumulation of lipofuscin-like material in RPE cells. PMID:24664687

  10. 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

  11. Lysosomal integral membrane protein-2: a new player in lysosome-related pathology.

    PubMed

    Gonzalez, Ashley; Valeiras, Mark; Sidransky, Ellen; Tayebi, Nahid

    2014-02-01

    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 disk 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

  12. 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

  13. Target Selection for the Apache Point Observatory Galactic Evolution Experiment (APOGEE)

    E-print Network

    Zasowski, G; Frinchaboy, P M; Majewski, S R; Nidever, D L; Pinto, H J Rocha; Girardi, L; Andrews, B; Chojnowski, S D; Cudworth, K M; Jackson, K; Munn, J; Skrutskie, M F; Beaton, R L; Blake, C H; Covey, K; Deshpande, R; Epstein, C; Fabbian, D; Fleming, S W; Hernandez, A Garcia; Herrero, A; Mahadevan, S; Meszaros, Sz; Schultheis, M; Sellgren, K; Terrien, R; van Saders, J; Prieto, C Allende; Bizyaev, D; Burton, A; Cunha, K; da Costa, L N; Hasselquist, S; Hearty, F; Holtzman, J; Perez, A E Garcia; Maia, M A G; O'Connell, R W; O'Donnell, C; Pinsonneault, M; Santiago, B X; Schiavon, R P; Shetrone, M; Smith, V; Wilson, J C

    2013-01-01

    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 ~100,000 red giant stars, selected to minimize observational biases in age and metallicity. We present the methodology and ...

  14. Lorcaserin and pimavanserin: emerging selectivity of serotonin receptor subtype–targeted 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 subtype–selective drugs, lorcaserin and pimavanserin, which target the 5HT2C and 5HT2A receptors and provide new treatments for obesity and Parkinson’s disease psychosis, respectively. PMID:24292660

  15. Targeted activation of silent natural product biosynthesis pathways by reporter-guided mutant selection.

    PubMed

    Guo, Fang; Xiang, Sihai; Li, Liyuan; Wang, Bin; Rajasärkkä, Johanna; Gröndahl-Yli-Hannuksela, Kirsi; Ai, Guomin; Metsä-Ketelä, Mikko; Yang, Keqian

    2015-03-01

    The continuously increasing genome sequencing data has revealed numerous cryptic pathways, which might encode novel secondary metabolites with interesting biological activities. However, utilization of this hidden potential has been hindered by the observation that many of these gene clusters remain silent (or poorly expressed) under laboratory conditions. Here we present reporter-guided mutant selection (RGMS) as an effective and widely applicable method for targeted activation of silent gene clusters in the native producers. The strategy takes advantage of genome-scale random mutagenesis for generation of genetic diversity and a reporter-guided selection system for the identification of the desired target-activated mutants. It was first validated in the re-activation of jadomycin biosynthesis in Streptomyces venezuelae ISP5230, where high efficiency of activation was achieved. The same strategy was then applied to a hitherto unactivable pga gene cluster in Streptomyces sp. PGA64 leading to the identification of two new anthraquinone aminoglycosides, gaudimycin D and E. PMID:25554073

  16. Enzymatic reduction of disulfide bonds in lysosomes: Characterization of a Gamma-interferon-inducible lysosomal thiol reductase (GILT)

    Microsoft Academic Search

    Balasubramanian Arunachalam; Uyen T. Phan; Hans J. Geuze; Peter Cresswell

    2000-01-01

    Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond

  17. Armed E-Bunny: a selective dynamic compiler for embedded Java virtual machine targeting ARM processors

    Microsoft Academic Search

    Mourad Debbabi; Azzam Mourad; Nadia Tawbi

    2005-01-01

    This paper presents a new selective dynamic compilation technique targeting ARM 16\\/32-bit embedded system processors. This compiler is built inside the J2ME\\/CLDC (Java 2 Micro Edition for Connected Limited Device Configuration) platform [8]. The primary objective of our work is to come up with an efficient, lightweight and low-footprint accelerated Java virtual machine ready to be executed on embedded machines.

  18. A selective dynamic compiler for embedded Java virtual machines targeting ARM processors

    Microsoft Academic Search

    Mourad Debbabi; Abdelouahed Gherbi; Azzam Mourad; Hamdi Yahyaoui

    2006-01-01

    This paper presents a new selective dynamic compilation technique targeting ARM 16\\/32-bit embedded system processors. This compiler is built inside the J2ME\\/CLDC (Java 2 Micro Edition for Connected Limited Device Configuration) platform [Sun MicroSystems, Java 2 Platform, Micro Edition, Version 1.0 Connected, Limited Device Configuration, Specification, Technical Report, Sun Microsystems, CA, USA, May 2000]. The primary objective of this work

  19. Global changes in STAT target selection and transcription regulation upon interferon treatments

    Microsoft Academic Search

    Stephen E. Hartman; Paul Bertone; Anjali K. Nath; Thomas E. Royce; Mark Gerstein; Sherman Weissman; Michael Snyder

    2008-01-01

    The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of

  20. Spectral selective radio frequency emissions from laser induced breakdown of target materials

    SciTech Connect

    Vinoth Kumar, L.; Manikanta, E.; Leela, Ch.; Prem Kiran, P., E-mail: premkiranuoh@gmail.com [Advanced Centre of Research in High Energy Materials (ACRHEM), University of Hyderabad, Hyderabad 500046 (India)

    2014-08-11

    The radio frequency emissions scanned over broad spectral range (30?MHz–1?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.

  1. Selection of RIB targets using ion implantation at the Holifield Radioactive Ion Beam Facility

    NASA Astrophysics Data System (ADS)

    Alton, G. D.; Dellwo, J.

    1996-02-01

    Among several major challenges posed by generating and accelerating adequate intensities of RIBs, selection of the most appropriate target material is perhaps the most difficult because of the requisite fast and selective thermal release of minute amounts of the short-lived product atoms from the ISOL target in the presence of bulk amounts of target material. Experimental studies are under way at the Oak Ridge National Laboratory (ORNL) which are designed to measure the time evolution of implanted elements diffused from refractory target materials which are candidates for forming radioactive ion beams (RIBs) at the Holifield Radioactive Ion Beam Facility (HRIBF). The diffusion coefficients are derived by comparing experimental data with numerical solutions to a one-dimensional form of Fick's second equation for ion implanted distributions. In this report, we describe the experimental arrangement, experimental procedures, and provide time release data and diffusion coefficients for releasing ion implanted 37Cl from Zr 5Si 3 and 75As, 79Br, and 78Se from Zr 5Ge 3 and estimates of the diffusion coefficients for 35Cl, 63Cu, 65Cu, 69Ga, and 71Ga diffused from BN; 35Cl, 63Cu, 65Cu, 69Ga, 75As, and 78Se diffused from C; 35Cl, 68Cu, 69Ga, 75As, and 78Se diffused from Ta.

  2. BioGPS: navigating biological space to predict polypharmacology, off-targeting, and selectivity.

    PubMed

    Siragusa, Lydia; Cross, Simon; Baroni, Massimo; Goracci, Laura; Cruciani, Gabriele

    2015-03-01

    The structural comparison of protein binding sites is increasingly important in drug design; identifying structurally similar sites can be useful for techniques such as drug repurposing, and also in a polypharmacological approach to deliberately affect multiple targets in a disease pathway, or to explain unwanted off-target effects. Once similar sites are identified, identifying local differences can aid in the design of selectivity. Such an approach moves away from the classical "one target one drug" approach and toward a wider systems biology paradigm. Here, we report a semiautomated approach, called BioGPS, that is based on the software FLAP which combines GRID Molecular Interactions Fields (MIFs) and pharmacophoric fingerprints. BioGPS comprises the automatic preparation of protein structure data, identification of binding sites, and subsequent comparison by aligning the sites and directly comparing the MIFs. Chemometric approaches are included to reduce the complexity of the resulting data on large datasets, enabling focus on the most relevant information. Individual site similarities can be analyzed in terms of their Pharmacophoric Interaction Field (PIF) similarity, and importantly the differences in their PIFs can be extracted. Here we describe the BioGPS approach, and demonstrate its applicability to rationalize off-target effects (ER? and SERCA), to classify protein families and explain polypharmacology (ABL1 kinase and NQO2), and to rationalize selectivity between subfamilies (MAP kinases p38?/ERK2 and PPAR?/PPAR?). The examples shown demonstrate a significant validation of the method and illustrate the effectiveness of the approach. PMID:25556939

  3. 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

  4. 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.

  5. Risk-targeted selection of agricultural holdings for post-epidemic surveillance: estimation of efficiency gains.

    PubMed

    Handel, Ian G; de C Bronsvoort, Barend M; Forbes, John F; Woolhouse, Mark E J

    2011-01-01

    Current post-epidemic sero-surveillance uses random selection of animal holdings. A better strategy may be to estimate the benefits gained by sampling each farm and use this to target selection. In this study we estimate the probability of undiscovered infection for sheep farms in Devon after the 2001 foot-and-mouth disease outbreak using the combination of a previously published model of daily infection risk and a simple model of probability of discovery of infection during the outbreak. This allows comparison of the system sensitivity (ability to detect infection in the area) of arbitrary, random sampling compared to risk-targeted selection across a full range of sampling budgets. We show that it is possible to achieve 95% system sensitivity by sampling, on average, 945 farms with random sampling and 184 farms with risk-targeted sampling. We also examine the effect of ordering samples by risk to expedite return to a disease-free status. Risk ordering the sampling process results in detection of positive farms, if present, 15.6 days sooner than with randomly ordered sampling, assuming 50 farms are tested per day. PMID:21674022

  6. The role of ubiquitination in lysosomal trafficking of ?-opioid receptors

    PubMed Central

    Henry, Anastasia G; White, Ian J.; Marsh, Mark; von Zastrow, Mark; Hislop, James N

    2010-01-01

    Lysyl-ubiquitination of signaling receptors is widely recognized to drive their proteolytic down-regulation via the multivesicular body (MVB) / lysosome pathway. Ubiquitination can act at multiple steps in this pathway, depending on receptor type and organism examined. No previous study has identified specific trafficking step(s) controlled by ubiquitination of a mammalian seven-transmembrane receptor (7TMR). The ?-opioid receptor (DOR) undergoes ligand-induced is a mammalian 7TMR down-regulation by ESCRT-dependent endocytic trafficking to lysosomes. In contrast to a number of other signaling receptors, the DOR can down-regulate effectively when its ubiquitination is prevented. We explored the membrane trafficking basis of this behavior. First, we show that that undergoes rapid lysosomal down-regulation physiologically, but this 7TMR has a still-unexplained ability to down-regulate effectively even when its ubiquitination is blocked. define pathway underlying internalized DORs traverse the canonical MVB pathway and localize to intralumenal vesicles (ILVs). Second, we show that DOR ubiquitination stimulates, but is not essential for, receptor transfer to ILVs and proteolysis of the receptor endodomain. Third, we show that receptor uHere we show that DORs traffic via morphologically typical MVBs and, similar to other signaling receptors, ubiquitination of DORs promotes the transfer of receptors from the limiting membrane of MVBs into intralumenal vesicles (ILVs). However, biquitination plays no detectable role in the early sorting of internalized DORs out of the recycling pathway. Finally, we show that DORs undergo extensive proteolytic fragmentation in the ectodomain, even when receptor ubiquitination is prevented or ILV formation itself is blocked. Together these results are sufficient to explain why DORs down-regulate effectively in the absence of ubiquitination, and they place a discrete molecular sorting operation in the MVB pathway effectively upstream of the ESCRT. selectively of without More generally, these findings support the hypothesis that unlike other signaling receptors presently described, this topological sorting function is regulatory rather than essential. Further, ubiquitination of DORs plays no detectable role in excluding internalized receptors from the bulk-recycling pathway. Together, these observations are sufficient to explain biochemical data indicating that ubiquitination of DORs produces a relatively subtle effect on the later digestion of receptor-derived proteolytic fragments. To our knowledge, this study provides the first systematic analysis of the role of ubiquitination in mediating lysosomal down-regulation of a mammalian 7TMR. This sbiochemically and functionally distinct mammalian cells can control the cytoplasmic accessibility of internalized signaling receptors independently from their ultimate trafficking fate. PMID:21106040

  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. Targeted delivery of photosensitizers: efficacy and selectivity issues revealed by multifunctional ORMOSIL nanovectors in cellular systems

    NASA Astrophysics Data System (ADS)

    Selvestrel, Francesco; Moret, Francesca; Segat, Daniela; Woodhams, Josephine H.; Fracasso, Giulio; Echevarria, Iria M. Rio; Baù, Luca; Rastrelli, Federico; Compagnin, Chiara; Reddi, Elena; Fedeli, Chiara; Papini, Emanuele; Tavano, Regina; MacKenzie, Alexandra; Bovis, Melissa; Yaghini, Elnaz; MacRobert, Alexander J.; Zanini, Silvia; Boscaini, Anita; Colombatti, Marco; Mancin, Fabrizio

    2013-06-01

    PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data suggest the interference of the stealth-conferring layer (PEG) with small targeting agents, but not with bulky antibodies. Moreover, we showed that selective photokilling of tumour cells may be limited even in the case of efficient targeting because of intrinsic transport limitations of active cellular uptake mechanisms or suboptimum localization.PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data suggest the interference of the stealth-conferring layer (PEG) with small targeting agents, but not with bulky antibodies. Moreover, we showed that selective photokilling of tumour cells may be limited even in the case of efficient targeting because of intrinsic transport limitations of active cellular uptake mechanisms or suboptimum localization. Electronic supplementary information (ESI) available: Experimental procedures and additional characterization of nanoparticles. See DOI: 10.1039/c3nr00402c

  9. Target Selection for the LBTI Hunt for Observable Signatures of Terrestrial Planetary Systems

    NASA Astrophysics Data System (ADS)

    Weinberger, Alycia J.; Roberge, A.; Kennedy, G.; Hinz, P.; Bryden, G.; Defrere, D.; Wyatt, M.; Stapelfeldt, K. R.; Rieke, G.; Danchi, W. C.; Mennesson, B.; Millan-Gabet, R.; Serabyn, G.; Skemer, A.; LBTI-HOSTS

    2014-01-01

    The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer (LBTI) will survey nearby stars for faint exozodiacal dust (exozodi). About 20% of field stars have cold debris disks created by the collisions and evaporation of planetesimals. Much less is known about warm circumstellar dust, such as that found in the vicinity of the Earth in our own system. This dust is generated in asteroidal collisions and cometary breakups, and current detection limits are at best ~500 times our system's level, i.e. 500 zodi. LBTI-HOSTS will be the first survey capable of measuring exozodi at the 10 zodi level (3?). Exozodi of this brightness would be the major source of astrophysical noise for a future space telescope aimed at direct imaging and spectroscopy of habitable zone terrestrial planets. Detections of warm dust will also reveal new information about planetary system architectures and evolution. We describe the target star selection by the LBTI Science Team to satisfy the goals of the HOSTS survey -- to fully inform target selection for a future exoEarth mission. We are interested in actual stars likely to be observed by a mission and stars whose observation will enable sensible extrapolations to those stars that cannot be observed. We integrated two approaches to generate the HOSTS target list. The mission-driven approach concentrates on F, G, and K-type stars that are the best targets for future direct observations of exoEarths, thereby providing model-independent “ground truth” dust observations. However, not every potential target of a future exoEarth mission can be observed with LBTI. The sensitivity-driven approach selects targets based only on what exozodi sensitivity could be achieved, without consideration of exoEarth mission constraints. This naturally selects more luminous stars (A and early F-type stars). In both cases, all stars are close enough to Earth such that their habitable zones are resolvable by LBTI and bright enough at N-band (10 ?m) to provide excellent sensitivity. We also discuss observational and astrophysical motivations for excluding binaries of certain separations.

  10. 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.

  11. 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

  12. 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.

  13. Diffusion properties of ion-implanted species in selected target materials

    NASA Astrophysics Data System (ADS)

    Alton, G. D.; Dellwo, J.; Carter, H. K.; Kormicki, J.; di Bartolo, G.; Batchelder, J. C.; Breitenbach, J.; Chediak, J. Alexander; Jentoff-Nilsen, K.; Ichikawa, S.

    1995-03-01

    Experiments designed to select the most appropriate target material for generating a particular radioactive ion beam (RIB) are in progress at the Oak Ridge National Laboratory. They are important to the future success of the Holifield Radioactive Ion Beam Facility (HRIBF). 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 `on-line' at the UNISOR facility. The intensity versus time of implanted species, which diffuse from the high-temperature target material (approximately equals 1700 degree(s)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 Zr5Si3 and As, Br, and Se implanted into and diffused from Zr5Ge3 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 also presented.

  14. Target-selective Phototherapy using a Ligand-based Photosensitizer for Type 2 Cannabinoid Receptor

    PubMed Central

    Zhang, Shaojuan; Jia, Ningyang; Shao, Pin; Tong, Qin; Xie, Xiangqun; Bai, Mingfeng

    2014-01-01

    SUMMARY Phototherapy is a powerful noninvasive treatment approach for cancer treatment, with several agents currently being in clinical use. Despite the progress and promise, most current phototherapy agents have serious side effects as they can lead to damage to healthy tissue, even when the photosensitizers is fused to targeting molecules, due to non-specific light activation of the unbound photosensitizer. To overcome these limitations, we develop a phototherapy agent that combines a functional ligand and a near infrared phthalocyanine dye. The target we focus on here is type 2 cannabinoid receptor (CB2R) which has been considered an attractive therapeutic target for phototherapy given it is over-expressed by many types of cancers which are located at a surface or can be reached by an endoscope. We show that our CB2R-targeted phototherapy agent, IR700DX-mbc94, is specific for CB2R and effective only when bound to the target receptor, suggesting a receptor-selective phototherapy mechanism and eliminating key side effects. Overall, this opens up opportunity for development of an alternative treatment option for CB2R positive cancers. PMID:24583052

  15. Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection

    PubMed Central

    Pattanayak, Vikram; Ramirez, Cherie L.; Joung, J. Keith; Liu, David R.

    2011-01-01

    Engineered zinc finger nucleases (ZFNs) are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Analysis of the identified sites in cultured human cells revealed CCR5-224-induced mutagenesis at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design. PMID:21822273

  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. Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing

    PubMed Central

    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, Campylobacer 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 providesd 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

  18. Dried blood spots for the enzymatic diagnosis of lysosomal storage diseases in dogs and cats

    PubMed Central

    Sewell, Adrian C.; Haskins, Mark E.; Giger, Urs

    2012-01-01

    Background In people lysosomal storage diseases (LSD) can be diagnosed by assaying enzyme activities in dried blood spots (DBS). Objective The aim of this study was to evaluate the feasibility of using DBS samples from dogs and cats to measure lysosomal enzymatic activities and diagnose LSD. Methods Drops of fresh whole blood collected in EDTA from dogs and cats with known or suspected LSD and from clinically healthy dogs and cats were placed on neonatal screening cards, dried, and mailed to the Metabolic Laboratory, University Children’s Hospital, Frankfurt, Germany. Activities of selected lysosomal enzymes were measured using fluorescent substrates in a 2-mm diameter disk (~2.6 ?L blood) punched from the DBS. Results were expressed as nmol substrate hydrolyzed per mL of blood per minute or hour. Results Reference values were established for several lysosomal enzyme activities in DBS from dogs and cats; for most enzymes, activities were higher than those published for human samples. Activities of ?-glucuronidase, N-acetylglucosamine-4-sulfatase (arylsulfatase B), ?-mannosidase, ?-galactosidase, ?-fucosidase, and hexosaminidase A were measureable in DBS from healthy cats and dogs; ?-iduronidase activity was measureable only in cats. In samples from animals with LSD, markedly reduced activity of a specific enzyme was found. In contrast, in samples from cats affected with mucolipidosis II activities of lysosomal enzymes were markedly increased. Conclusions Measurement of lysosomal enzyme activities in DBS provides an inexpensive, simple, and convenient method to screen animals for suspected LSD and requires only a small sample volume. For diseases in which the relevant enzyme activity can be measured in DBS, a specific diagnosis can be made. PMID:23121383

  19. Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

    PubMed Central

    López 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

  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. Lysosomal Destabilization Contributes to Apoptosis of Germinal Center B-lymphocytes

    Microsoft Academic Search

    Kirsten van Nierop; Femke J. M. Muller; Jan Stap; Cornelis J. F. Van Noorden; Marco van Eijk; Cornelis de Groot

    2006-01-01

    During germinal center (GC) reactions, B-lymphocytes with high-affinity B-cell receptors are selected. Regulation of apoptosis is a key process in selecting such wanted B-cells and in eliminating B-cells with unwanted specificities. In this paper, we show that apoptosis in human GC B-cells involves lysosomal destabilization, which is strictly controlled by caspase-8 activity, but not by caspase-9 activity. Ligation of CD40

  2. 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

  3. Target Selection for the LBTI Hunt for Observable Signatures of Terrestrial Planetary Systems

    NASA Astrophysics Data System (ADS)

    Roberge, A.; Weinberger, A.; Kennedy, G.; Defrère, D.; LBTI Instrument; Science Teams

    2014-03-01

    The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer (LBTI) will survey nearby stars for faint exozodiacal dust (exozodi). This warm circumstellar dust, analogous to the interplanetary dust found in the vicinity of the Earth in our own system, is produced in comet breakups and asteroid collisions. Exozodi will be the major source of astrophysical noise for a future space telescope aimed at direct imaging and spectroscopy of habitable zone terrestrial planets (exo-Earths). About 20% of nearby field stars have cold dust coming from planetesimals at large distances from the stars (Eiroa et al. 2013). Much less is known about exozodi; current detection limits for individual stars are at best ~ 500 times our solar system's level (aka. 500 zodi). LBTI-HOSTS will be the first survey capable of measuring exozodi at the 10 zodi level (3s). Detections of warm dust will also reveal new information about planetary system architectures and evolution. We describe the target star selection by the LBTI Science Team to satisfy the goals of the HOSTS survey - to inform mission design and target selection for a future exo-Earth mission. We are interested in both 1) actual stars likely to be observed by such a mission and 2) stars whose observation will enable sensible extrapolations for stars that cannot be observed with LBTI. We integrated two approaches to generate the HOSTS target list. The mission-driven approach concentrates on F, G, and K-type stars that are the best targets for future direct observations of exo-Earths, thereby providing model-independent "ground truth" dust observations. However, not every potential target of a future exo-Earth mission can be observed with LBTI. The sensitivity-driven approach selects targets based on maximizing the exozodi sensitivity that can be achieved, without consideration of exo-Earth mission constraints. This naturally chooses more luminous stars (A and early F-type stars). In both cases, all stars are close enough to Earth such that their habitable zones are resolvable by LBTI and bright enough at N-band (10 µm) to provide excellent sensitivity. We also discuss observational and astrophysical motivations for excluding binaries of certain separations.

  4. Gold nanorods for target selective SPECT/CT imaging and photothermal therapy in vivo.

    PubMed

    Jang, Boseung; Park, Seonhwa; Kang, Se Hun; Kim, Joa Kyum; Kim, Seok-Ki; Kim, In-Hoo; Choi, Yongdoo

    2012-03-01

    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, H(2)N-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

  5. 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

  6. Body condition score as a selection tool for targeted selective treatment-based nematode control strategies in Merino ewes.

    PubMed

    Cornelius, M P; Jacobson, C; Besier, R B

    2014-12-15

    Sheep nematode control utilising refugia-based strategies have been shown to delay anthelmintic resistance, but the optimal indices to select individuals to be left untreated under extensive sheep grazing conditions are not clear. This experiment tested the hypothesis that high body condition can indicate ability of mature sheep to better cope with worms and therefore remain untreated in a targeted treatment programme. Adult Merino ewes from flocks on two private farms located in south-west Western Australia (Farm A, n = 271, and Farm B, n = 258) were measured for body condition score (BCS), body weight and worm egg counts (WEC) on four occasions between May and December (pre-lambing, lamb marking, lamb weaning and post-weaning). Half of the ewes in each flock received anthelmintic treatments to suppress WEC over the experimental period and half remained untreated (unless critical limits were reached). Response to treatment was analysed in terms of BCS change and percentage live weight change. No effect of high or low initial WEC groups was shown for BCS response, and liveweight responses were inconsistent. A relatively greater BCS response to treatment was observed in ewes in low BCS pre-lambing compared to better-conditioned ewes on one farm where nutrition was sub-optimal and worm burdens were high. Sheep in low body condition pre-lambing were more than three times more likely to fall into a critically low BCS (<2.0) if left untreated. Recommendations can be made to treat ewes in lower BCS and leave a proportion of the higher body condition sheep untreated in a targeted selective treatment programme, to provide a population of non-resistant worms to delay the development of resistance. PMID:25466620

  7. Therapeutic approaches for neuronopathic lysosomal storage disorders

    Microsoft Academic Search

    Raphael Schiffmann

    2010-01-01

    Therapy of the central nervous system (CNS) manifestations of lysosomal storage diseases (LSDs) has remained a major challenge\\u000a because of its inability to deliver therapeutic agents efficiently across the intact blood–brain barrier. Non-specific therapies\\u000a such as hematopoietic stem cell transplantation have been useful in globoid cell leukodystrophy (Krabbe disease) and in some\\u000a mucopolysaccharidoses. Anti-inflammatory agents also show promise as adjuvant

  8. Genetic Counseling for Lysosomal Storage Diseases

    Microsoft Academic Search

    Erin O'Rourke; Dawn Laney; Cindy Morgan; Kim Mooney; Jennifer Sullivan

    In many ways, the role of the genetic counselor working with patients and families with a lysosomal storage disease is similar\\u000a to a counselor in other pediatric and adult counseling situations. The goals of counseling; education, access to health care,\\u000a and supportive counseling are the same. Although the goals of counseling are simply stated, an effective counseling session\\u000a is always

  9. Lysosomes in iron metabolism, ageing and apoptosis

    Microsoft Academic Search

    Tino Kurz; Alexei Terman; Bertil Gustafsson; Ulf T. Brunk

    2008-01-01

    The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived\\u000a proteins and all organelles. The compartment consists of numerous acidic vesicles (pH ?4 to 5) that constantly fuse and divide.\\u000a It receives a large number of hydrolases (?50) from the trans-Golgi network, and substrates from both the cells’ outside (heterophagy) and inside

  10. A Flow Cytometric Assay for Lysosomal Glucocerebrosidase

    Microsoft Academic Search

    Helmuth H. G. van Es; Marlon Veldwijk; Menzo Havenga; Dinko Valerio

    1997-01-01

    A flow cytometric assay is described for the determination of glucocerebrosidase (GC) activity using fluorescein di-?-glucopyranoside (FDGlu). Fluorescent product is formed upon intracellular hydrolysis of FDGlu and is measured in the FL1 channel of a flow cytometer. We show that the assay is specific for lysosomal ?-glucosidase or glucocerebrosidase (1) by concentration-dependent inhibition of GC activity by conditurol-?-epoxide (CBE), a

  11. The anticancer effect of cytotoxin 1 from Naja atra Cantor venom is mediated by a lysosomal cell death pathway involving lysosomal membrane permeabilization and cathepsin B release.

    PubMed

    Wu, Minyan; Ming, Wei; Tang, Ya; Zhou, Shengming; Kong, Tianhan; Dong, Weihua

    2013-01-01

    The cytotoxin family of cobra venom proteins, also called cardiotoxins, can activate both necrotic and apoptotic cell death pathways in cancer cells. Cytotoxin 1 (CTX1)from Naja atra Cantor venom is a 60 amino acid, 6698 Da protein with as yet untested anticancer efficacy and cell selectivity. We tested the toxicity of CTX1 on a number of cancer cell lines (MCF-7, P388, K562, and H22) and on one normal human cell line (16HBE). The rank order of cytotoxicity was MCF-7 > P388 ? K562 >H22 ? 16HBE, indicating that the effect of CTX1 on certain cancer cell types was relatively selective.Treatment with CTX1 greatly prolonged the survival of P388 ascites tumors bearing KM mice compared to cyclophosphamide treatment. Cell viability, apoptosis, and lysosomal permeability assays all demonstrated that CTX1 induced dose- and time-dependent cell death, with most cells exhibiting the morphological and biochemical features of late apoptosis and necrosis. Mitochondrial membrane potential was lost in CTX1-treated P388 cells. In addition, CTX1 induced an increase in both lysosomal membrane permeability and cathepsin B protease activity. These analyses reveal that CTX1 possesses significant and selective anticancer activity, likely by inducing programmed cell death through mitochondrial and/or lysosomal pathways. PMID:23711147

  12. 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

  13. The transcription factor NRSF contributes to epileptogenesis by selective repression of a subset of target genes.

    PubMed

    McClelland, Shawn; Brennan, Gary P; Dubé, Celine; Rajpara, Seeta; Iyer, Shruti; Richichi, Cristina; Bernard, Christophe; Baram, Tallie Z

    2014-01-01

    The mechanisms generating epileptic neuronal networks following insults such as severe seizures are unknown. We have previously shown that interfering with the function of the neuron-restrictive silencer factor (NRSF/REST), an important transcription factor that influences neuronal phenotype, attenuated development of this disorder. In this study, we found that epilepsy-provoking seizures increased the low NRSF levels in mature hippocampus several fold yet surprisingly, provoked repression of only a subset (?10%) of potential NRSF target genes. Accordingly, the repressed gene-set was rescued when NRSF binding to chromatin was blocked. Unexpectedly, genes selectively repressed by NRSF had mid-range binding frequencies to the repressor, a property that rendered them sensitive to moderate fluctuations of NRSF levels. Genes selectively regulated by NRSF during epileptogenesis coded for ion channels, receptors, and other crucial contributors to neuronal function. Thus, dynamic, selective regulation of NRSF target genes may play a role in influencing neuronal properties in pathological and physiological contexts. PMID:25117540

  14. Trihydroxamate Siderophore-Fluoroquinolone Conjugates are Selective Sideromycin Antibiotics that Target Staphylococcus aureus

    PubMed Central

    Wencewicz, Timothy A.; Long, Timothy E.; Möllmann, Ute; Miller, Marvin J.

    2013-01-01

    Siderophores are multidentate iron(III) chelators used by bacteria for iron assimilation. Sideromycins, also called siderophore-antibiotic conjugates, are a unique subset of siderophores that enter bacterial cells via siderophore uptake pathways and deliver the toxic antibiotic in a ‘Trojan Horse’ fashion. Sideromycins represent a novel antibiotic delivery technology with untapped potential for developing sophisticated microbe-selective antibacterial agents that limit the emergence of bacterial resistance. The chemical synthesis of a series of mono-, bis-, and trihydroxamate sideromycins are described here along with their biological evaluation in antibacterial susceptibility assays. The linear hydroxamate siderophores used for the sideromycins in this study were derived from the ferrioxamine family and inspired by the naturally occurring salmycin sideromycins. The antibacterial agents used were a ?-lactam carbacepholosporin, Lorabid®, and a fluoroquinolone, ciprofloxacin, chosen for the different locations of their biological targets, the periplasm (extracellular) and the cytoplasm (intracellular). The linear hydroxamate-based sideromycins were selectively toxic towards Gram-positive bacteria, especially Staphylococcus aureus SG511 (MIC = 1.0 µM for the trihydroxamate-fluoroquinolone sideromycin). Siderophore-sideromycin competition assays demonstrated that only the fluoroquinolone sideromycins required membrane transport to reach their cytoplasmic biological target and that a trihydroxamate siderophore backbone was required for protein-mediated active transport of the sideromycins into S. aureus cells via siderophore uptake pathways. This work represents a comprehensive study of linear hydroxamate sideromycins and teaches how to build effective hydroxamate-based sideromycins as Gram-positive selective antibiotic agents. PMID:23350642

  15. Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene

    SciTech Connect

    Carrer, H.; Maliga, P. [State Univ. of New Jersey, Piscataway, NJ (United States)] [State Univ. of New Jersey, Piscataway, NJ (United States)

    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.

  16. 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

    2014-11-20

    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

  17. Targeting Class IA PI3K Isoforms Selectively Impairs Cell Growth, Survival, and Migration in Glioblastoma

    PubMed Central

    Höland, Katrin; Boller, Danielle; Hagel, Christian; Dolski, Silvia; Treszl, András; Pardo, Olivier E.; ?wiek, Paulina; Salm, Fabiana; Leni, Zaira; Shepherd, Peter R.; Styp-Rekowska, Beata; Djonov, Valentin; von Bueren, André O.; Frei, Karl; Arcaro, Alexandre

    2014-01-01

    The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110? was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110? expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110?/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110? activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110? or PI3K p110? also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110? did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110? can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110?/p-S6. PMID:24718026

  18. 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

  19. 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

  20. Targeting a cryptic allosteric site for selective inhibition of the oncogenic protein tyrosine phosphatase Shp2.

    PubMed

    Chio, Cynthia M; Lim, Christopher S; Bishop, Anthony C

    2015-01-20

    Protein tyrosine phosphatases (PTPs) have been the subject of considerable pharmaceutical-design efforts because of the ubiquitous connections between misregulation of PTP activity and human disease. PTP-inhibitor discovery has been hampered, however, by the difficulty in identifying cell-permeable compounds that can selectively target PTP active sites, and no PTP inhibitors have progressed to the clinic. The identification of allosteric sites on target PTPs therefore represents a potentially attractive solution to the druggability problem of PTPs. Here we report that the oncogenic PTP Shp2 contains an allosteric-inhibition site that renders the enzyme sensitive to potent and selective inhibition by cell-permeable biarsenical compounds. Because Shp2 contains no canonical tetracysteine biarsenical-binding motif, the enzyme's inhibitor-binding site is not readily predictable from its primary or three-dimensional structure. Intriguingly, however, Shp2's PTP domain does contain a cysteine residue (C333) at a position that is removed from the active site and is occupied by proline in other classical PTPs. We show that Shp2's unusual cysteine residue constitutes part of a Shp2-specific allosteric-inhibition site, and that Shp2's sensitivity to biarsenicals is dependent on the presence of the naturally occurring C333. The determinative role of this residue in conferring inhibitor sensitivity is surprising because C333's side chain is inaccessible to solvent in Shp2 crystal structures. The discovery of this cryptic Shp2 allosteric site may provide a means for targeting Shp2 activity with high specificity and suggests that buried-yet-targetable allosteric sites could be similarly uncovered in other protein families. PMID:25519989

  1. Purification and biochemical characterization of a lysosomal ?-fucosidase from the deuterostomia Asterias rubens.

    PubMed

    Visa, Merino; Hammer, Elke; Völker, Uwe; Koliwer-Brandl, Hendrik; Kelm, Sørge; Nadimpalli, Siva Kumar

    2012-05-01

    In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of ?-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ?56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC-MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates. PMID:22366030

  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; d’Azzo, Alessandra

    2013-01-01

    Alzheimer’s 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. Targeted Quantitation of Acetylated Lysine Peptides by Selected Reaction Monitoring Mass Spectrometry

    PubMed Central

    Rardin, Matthew J.; Held, Jason M.; Gibson, Bradford W.

    2015-01-01

    Mass spectrometry (MS) allows for the large-scale identification of multiple peptide analytes in complex mixtures. However, the low abundance of acetylated peptides in the overall mixture requires an enrichment step. After enrichment, the resulting acetylated peptides of interest can be quantitated using selected reaction monitoring (SRM)-MS with stable isotope dilution. Here, we describe the enrichment of lysine acetylated peptides from typsin digested mouse liver mitochondria, and the targeted quantitation of a known lysine acetylation site in succinate dehydrogenase A using SRM-MS on a triple quadrupole instrument. PMID:24014403

  4. The RNA Polymerase II Trigger Loop Functions in Substrate Selection and is Directly Targeted by ?-amanitin

    PubMed Central

    Kaplan, Craig D.; Larsson, Karl-Magnus; Kornberg, Roger D.

    2008-01-01

    Summary Structural, biochemical and genetic studies have led to proposals that a mobile element of multi-subunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by ?-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to ?-amanitin, indicating a functional interaction between His1085 and ?-amanitin that is supported by re-refinement of an ?-amanitin-Pol II crystal structure. We propose that ?-amanitin inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct ?-amanitin interference with the TL. PMID:18538653

  5. Plasma chitotriosidase activity in children with lysosomal storage disorders.

    PubMed

    Sheth, Jayesh J; Sheth, Frenny J; Oza, Nrupesh J; Gambhir, Prakash S; Dave, Usha P; Shah, Raju C

    2010-02-01

    Chitotriosidase (ChT) is an enzyme that is selectively activated in tissue macrophage. This property of ChT makes it a potential marker for many disease process and prognostication. Present study has been carried out to know the significance of ChT as a screening marker in lysosomal storage disorders (LSDs) where tissue macrophage activation is commonly observed due to accumulation of substrate in various organs of the body. Study comprises of 20 healthy children in the age range of 10 days to 5 yrs and 56 children in the age range of 2.5 months to 13 yrs with regression of milestones, skeletal dysplasia, neuroregression and hepatosplenomegaly were selected for plasma ChT who had confirmed LSDs as carried out by specific lysosomal enzyme study from the leukocytes or fibroblasts. Plasma ChT was 55.21 +/- 20.81 nmol/ml/hr in twenty healthy age matched controls. Plasma ChT level was 42.88 to 79.78 nmol/ml/hr in thirteen of 56 (23.21%) children with LSDs like Morquio-B, Pompe, Metachromatic leucodystrophy (MLD), Sandhoff and Niemann-Pick disease type C (NPD-C). While in 43 (76.78%) children it was in the range of 213.74 to 23,511.40 nmol/ml/hr. who had LSDs like Morquio-B, Sly syndrome, MLD, GM2 Gangliosidosis, NPD-A/B and Gaucher disease (GD). Marked elevated ChT (4,000 to 23,511 nmol/ml/hr) was observed in all cases of GD (n=7) and NDP-A/B. It can be concluded from the present study that moderately raised activity of ChT can be utilized as a positive predictive test for certain LSD's. Those with marked elevated ChT have confirmed GD or NPD-A/B making it a strong screening marker for this group of diseases. PMID:19936666

  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. Pulmonary arterial hypertension associated with impaired lysosomal endothelin-1 degradation.

    PubMed

    Recla, Sabine; Hahn, Andreas; Apitz, Christian

    2015-04-01

    We report on a boy with severe pulmonary arterial hypertension associated with mucolipidosis, a rare lysosomal storage disorder. During diagnostic catheterisation, we found increased endothelin-1 levels, but normal big endothelin-1-levels (the precursor form of endothelin-1), which suggests impaired degradation of endothelin-1 rather than increased synthesis. As endothelin-1 degradation takes place in the lysosome, it appears likely that lysosomal dysfunction caused by the underlying disease contributes to the development of pulmonary arterial hypertension in this patient. PMID:24910177

  8. 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

  9. SUMOylation modulates the transcriptional activity of androgen receptor in a target gene and pathway selective manner

    PubMed Central

    Sutinen, Päivi; Malinen, Marjo; Heikkinen, Sami; Palvimo, Jorma J.

    2014-01-01

    Androgen receptor (AR) plays an important regulatory role in prostate cancer. AR's transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates AR's interaction with the chromatin and the receptor's target gene selection. PMID:24981513

  10. 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.

  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. Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

    PubMed

    Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

    2012-02-01

    Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents. PMID:22064367

  13. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts

    PubMed Central

    Ginestier, Christophe; Liu, Suling; Diebel, Mark E.; Korkaya, Hasan; Luo, Ming; Brown, Marty; Wicinski, Julien; Cabaud, Olivier; Charafe-Jauffret, Emmanuelle; Birnbaum, Daniel; Guan, Jun-Lin; Dontu, Gabriela; Wicha, Max S.

    2010-01-01

    Recent evidence suggests that breast cancer and other solid tumors possess a rare population of cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. We report here the development of a strategy to target these breast cancer stem cells (CSCs) through blockade of the IL-8 receptor CXCR1. CXCR1 blockade using either a CXCR1-specific blocking antibody or repertaxin, a small-molecule CXCR1 inhibitor, selectively depleted the CSC population in 2 human breast cancer cell lines in vitro. Furthermore, this was followed by the induction of massive apoptosis in the bulk tumor population via FASL/FAS signaling. The effects of CXCR1 blockade on CSC viability and on FASL production were mediated by the FAK/AKT/FOXO3A pathway. In addition, repertaxin was able to specifically target the CSC population in human breast cancer xenografts, retarding tumor growth and reducing metastasis. Our data therefore suggest that CXCR1 blockade may provide a novel means of targeting and eliminating breast CSCs. PMID:20051626

  14. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts.

    PubMed

    Ginestier, Christophe; Liu, Suling; Diebel, Mark E; Korkaya, Hasan; Luo, Ming; Brown, Marty; Wicinski, Julien; Cabaud, Olivier; Charafe-Jauffret, Emmanuelle; Birnbaum, Daniel; Guan, Jun-Lin; Dontu, Gabriela; Wicha, Max S

    2010-02-01

    Recent evidence suggests that breast cancer and other solid tumors possess a rare population of cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. We report here the development of a strategy to target these breast cancer stem cells (CSCs) through blockade of the IL-8 receptor CXCR1. CXCR1 blockade using either a CXCR1-specific blocking antibody or repertaxin, a small-molecule CXCR1 inhibitor, selectively depleted the CSC population in 2 human breast cancer cell lines in vitro. Furthermore, this was followed by the induction of massive apoptosis in the bulk tumor population via FASL/FAS signaling. The effects of CXCR1 blockade on CSC viability and on FASL production were mediated by the FAK/AKT/FOXO3A pathway. In addition, repertaxin was able to specifically target the CSC population in human breast cancer xenografts, retarding tumor growth and reducing metastasis. Our data therefore suggest that CXCR1 blockade may provide a novel means of targeting and eliminating breast CSCs. PMID:20051626

  15. 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

  16. 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.

  17. 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.

  18. Linking Albinism and Immunity: The Secrets of Secretory Lysosomes

    NSDL National Science Digital Library

    Jane Stinchcombe (Sir William Dunn School of Pathology; )

    2004-07-02

    Lysosomes are membrane-bound organelles that are found in all mammalian cells and contain hydrolases and lipases required for protein and membrane degradation. In many cells of the immune system, lysosomes also contain secretory proteins that can be released by regulated exocytosis in response to an external stimulus, providing different cell types with a wide range of effector functions. Melanosomes also use a lysosome-related organelle to secrete melanin for pigmentation. Links between albinism and immunity in patients have uncovered a number of key proteins required for lysosomal secretion and have revealed a versatile secretory mechanism that can be fine-tuned by distinct interactions in different cell types.

  19. Target dependence of orientation and direction selectivity of corticocortical projection neurons in the mouse V1

    PubMed Central

    Matsui, Teppei; Ohki, Kenichi

    2013-01-01

    Higher order visual areas that receive input from the primary visual cortex (V1) are specialized for the processing of distinct features of visual information. However, it is still incompletely understood how this functional specialization is acquired. Here we used in vivo two photon calcium imaging in the mouse visual cortex to investigate whether this functional distinction exists at as early as the level of projections from V1 to two higher order visual areas, AL and LM. Specifically, we examined whether sharpness of orientation and direction selectivity and optimal spatial and temporal frequency of projection neurons from V1 to higher order visual areas match with that of target areas. We found that the V1 input to higher order visual areas were indeed functionally distinct: AL preferentially received inputs from V1 that were more orientation and direction selective and tuned for lower spatial frequency compared to projection of V1 to LM, consistent with functional differences between AL and LM. The present findings suggest that selective projections from V1 to higher order visual areas initiates parallel processing of sensory information in the visual cortical network. PMID:24068987

  20. Hyperspectral band selection based on the aggregation of proximity measures for automated target detection

    NASA Astrophysics Data System (ADS)

    Ball, John E.; Anderson, Derek T.; Samiappan, Sathish

    2014-06-01

    Band selection is an important unsolved challenge in hyperspectral image processing that has been used for dimensionality reduction and classification improvement. To date, numerous researchers have investigated the unsupervised selection of band groups using measures such as correlation and Kullback-Leibler divergence. However, no clear winner has emerged across data sets and detection tasks. Herein, we investigate the utility of aggregating different proximity measures for band group selection. Specifically, we employ the Choquet integral with respect to different measures (capacities) as it is able to yield a variety of aggregation functions like t-norms, t-conorms and averaging operators. We explore the utility of aggregation in the context of single band, single band group, band group dimensionality reduction and multiple band group combinations in conjunction with support vector machine (SVM) based classification. Our preliminary experiments indicate there is value in aggregating different proximity measures. In some instances an intersection operator works well while in other cases a union operator is best. As may be expected, this can, and does vary per detection task. We also see that depending on the difficulty of the target detection problem, different aggregation, band grouping and combination strategies prevail. Advantages of our approach include; flexibility, the aggregation operator can be learned, and the method can default to a single proximity measure if needed and result, in the worst case, in no performance loss. Experiments are performed on three hyperspectral benchmark data sets to demonstrate the applicability of the proposed concepts.

  1. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs. PMID:24841365

  2. Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells

    Microsoft Academic Search

    Penelope A. Colbaugh; Margaret Stookey; Rockford K. Draper

    1989-01-01

    Abstract. We describe here the properties of a mutant of Chinese hamster,ovary cells that expresses a conditional-lethal mutation,affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation,group,of temperature-sensitive mu- tants selected for resistance to protein toxins (Col- baugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499-507).

  3. A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis

    PubMed Central

    Biery, Matthew C.; Stewart, Fiona J.; Stellwagen, Anne E.; Raleigh, Elisabeth A.; Craig, Nancy L.

    2000-01-01

    A robust Tn7-based in vitro transposition system is described that displays little target site selectivity, allowing the efficient recovery of many different transposon insertions in target DNAs ranging from small plasmids to cosmids to whole genomes. Two miniTn7 derivatives are described that are useful for the analysis of genes: one a derivative for making translational and transcriptional target gene fusions and the other a derivative that can generate 15 bp (5 amino acid) insertions in target DNAs (proteins). PMID:10666445

  4. Protective Effect of Enterosgel on Rat Liver lysosomes during Cytostatic treatment

    Microsoft Academic Search

    O. R. Grek; S. V. Mishenina; A. B. Pupyshev

    2002-01-01

    Polychemotherapy with a complex of cytostatics (cyclophosphamide, doxorubicin, vincristine, prednisolone) induces progressive damage to hepatocyte membranes, which manifested in labilization of lysosomes and activation of lysosomal enzymes and serum transaminases. Enterosgel stabilized liver lysosomes and reduced manifestation of hepatocyte cytolysis.

  5. Effective heritability of targets of sex-ratio selection under environmental sex determination.

    PubMed

    McGaugh, S E; Janzen, F J

    2011-04-01

    Selection is expected to maintain primary sex ratios at an evolutionary equilibrium. In organisms with temperature-dependent sex determination (TSD), targets of sex-ratio selection include the thermal sensitivity of the sex-determining pathway (hereafter, sex determination threshold) and nest-site choice. However, offspring sex may be canalized for nests located in thermally extreme environments; thus, genetic variance for the sex determination threshold is not expressed and is invisible to direct selection. The concept of 'effective heritability' accounts for this dependence and provides a more realistic prediction of the expected evolutionary response to selection in the wild. Past estimates of effective heritability of the sex determination threshold, which were derived from laboratory data, suggested that the potential for the sex determination threshold to evolve in the wild was extremely low. We re-evaluated original estimates of this parameter by analysing field-collected measures of nest temperatures, vegetation cover and clutch sex ratios from nests in a population of painted turtles (Chrysemys picta). We coupled these data with measurements of broad-sense heritability of the sex determination threshold in C. picta, using an experiment that splits clutches of eggs between a constant temperature (i.e. typical laboratory incubation) and a daily fluctuating temperature (i.e. similar to natural nests) with the same mean. We found that (i) the effective heritability of the sex determination threshold appears to have been historically underestimated and the effective heritability of nest-site choice has been overestimated and (ii) significant family-by-incubation treatment interaction exists for sex for C. picta between constant- and fluctuating-temperature regimes. Our results suggest that the thermal sensitivity of the sex-determining pathway may play a larger, more complex role in the microevolution of TSD than traditionally thought. PMID:21261771

  6. Targeting human T cells by retroviral vectors displaying antibody domains selected from a phage display library.

    PubMed

    Engelstädter, M; Bobkova, M; Baier, M; Stitz, J; Holtkamp, N; Chu, T H; Kurth, R; Dornburg, R; Buchholz, C J; Cichutek, K

    2000-01-20

    To generate T cell-specific retroviral vectors an scFv phage display library derived from immunized mice was selected for binding to the human T cell line Molt-4/8. The scFv cDNAs recovered from the selected phages were transiently expressed as an N-terminal fusion of the spleen necrosis virus (SNV) transmembrane protein (TM) subunit of the viral envelope protein (Env) in the cell line DSH-cxl, which packages the beta-galactosidase gene into SNV particles. Screening of supernatants from about 150 transfections resulted in the identification of 5 scFvs that mediated efficient transduction of Molt-4/8 cells. Using stable packaging cell lines vector preparations with titers greater than 10(4) EFU/ml on human T cells were obtained. The scFv 7A5 in particular was able to mediate selective transduction of human T cells with high efficiency. Titers of up to 106 EFU/ml were reached on Molt-4/8, Jurkat, and A301 cells, while titers on HeLa cells, TE671 cells, 293T cells, and HT1080 cells were below 102 EFU/ml. Transduction of stimulated primary human peripheral blood cells, which consisted mainly of T cells, was about fivefold more efficient than transduction of B cells. Western blot analysis of supernatant from the 7A5 packaging cells demonstrated incorporation of 7A5-TM into vector particles and indicated proteolytic processing of the coexpressed unmodified TM during particle formation. Binding of bacterially expressed 7A5-scFv to a panel of cell lines correlated well with the transduction results. These data provide the first proof of concept that a general approach can be taken to obtain scFvs able to mediate selective gene transfer into target cells. PMID:10680843

  7. Cold atmospheric plasma treatment selectively targets head and neck squamous cell carcinoma cells

    PubMed Central

    GUERRERO-PRESTON, RAFAEL; OGAWA, TAKENORI; UEMURA, MAMORU; SHUMULINSKY, GARY; VALLE, BLANCA L.; PIRINI, FRANCESCA; RAVI, RAJANI; SIDRANSKY, DAVID; KEIDAR, MICHAEL; TRINK, BARRY

    2014-01-01

    The treatment of locoregional recurrence (LRR) of head and neck squamous cell carcinoma (HNSCC) often requires a combination of surgery, radiation therapy and/or chemotherapy. Survival outcomes are poor and the treatment outcomes are morbid. Cold atmospheric plasma (CAP) is an ionized gas produced at room temperature under laboratory conditions. We have previously demonstrated that treatment with a CAP jet device selectively targets cancer cells using in vitro melanoma and in vivo bladder cancer models. In the present study, we wished to examine CAP selectivity in HNSCC in vitro models, and to explore its potential for use as a minimally invasive surgical approach that allows for specific cancer cell or tumor tissue ablation without affecting the surrounding healthy cells and tissues. Four HNSCC cell lines (JHU-022, JHU-028, JHU-029, SCC25) and 2 normal oral cavity epithelial cell lines (OKF6 and NOKsi) were subjected to cold plasma treatment for durations of 10, 30 and 45 sec, and a helium flow of 20 l/min?1 for 10 sec was used as a positive treatment control. We showed that cold plasma selectively diminished HNSCC cell viability in a dose-response manner, as evidenced by MTT assays; the viability of the OKF6 cells was not affected by the cold plasma. The results of colony formation assays also revealed a cell-specific response to cold plasma application. Western blot analysis did not provide evidence that the cleavage of PARP occurred following cold plasma treatment. In conclusion, our results suggest that cold plasma application selectively impairs HNSCC cell lines through non-apoptotic mechanisms, while having a minimal effect on normal oral cavity epithelial cell lines. PMID:25050490

  8. New Molecules and Old Drugs as Emerging Approaches to Selectively Target Human Glioblastoma Cancer Stem Cells

    PubMed Central

    Würth, Roberto; Barbieri, Federica; Florio, Tullio

    2014-01-01

    Despite relevant progress obtained by multimodal treatment, glioblastoma (GBM), the most aggressive primary brain tumor, is still incurable. The most encouraging advancement of GBM drug research derives from the identification of cancer stem cells (CSCs), since these cells appear to represent the determinants of resistance to current standard therapies. The goal of most ongoing studies is to identify drugs able to affect CSCs biology, either inducing selective toxicity or differentiating this tumor cell population into nontumorigenic cells. Moreover, the therapeutic approach for GBM could be improved interfering with chemo- or radioresistance mechanisms, microenvironment signals, and the neoangiogenic process. During the last years, molecular targeted compounds such as sorafenib and old drugs, like metformin, displayed interesting efficacy in preclinical studies towards several tumors, including GBM, preferentially affecting CSC viability. In this review, the latest experimental results, controversies, and prospective application concerning these promising anticancer drugs will be discussed. PMID:24527434

  9. Target selection for a hypervelocity asteroid intercept vehicle flight validation mission

    NASA Astrophysics Data System (ADS)

    Wagner, Sam; Wie, Bong; Barbee, Brent W.

    2015-02-01

    Asteroids and comets have collided with the Earth in the past and will do so again in the future. Throughout Earth's history these collisions have played a significant role in shaping Earth's biological and geological histories. The planetary defense community has been examining a variety of options for mitigating the impact threat of asteroids and comets that approach or cross Earth's orbit, known as near-Earth objects (NEOs). This paper discusses the preliminary study results of selecting small (100-m class) NEO targets and mission analysis and design trade-offs for validating the effectiveness of a Hypervelocity Asteroid Intercept Vehicle (HAIV) concept, currently being investigated for a NIAC (NASA Advanced Innovative Concepts) Phase 2 study. In particular this paper will focus on the mission analysis and design for single spacecraft direct impact trajectories, as well as several mission types that enable a secondary rendezvous spacecraft to observe the HAIV impact and evaluate it's effectiveness.

  10. Plausible improvements for selective targeting of dopamine receptors in therapy of Parkinson's disease.

    PubMed

    Luthra, Pratibha Mehta; Kumar, J B Senthil

    2012-12-01

    Parkinson's disease (PD) is a neurodegenerative condition characterized by progressive and profound loss of dopaminergic neurons in the substantia nigra pars compacta leading to the formation of eosinophillic, intracytoplamic, proteinacious inclusions termed as lewy bodies. L-dopa remains as a gold standard for the treatment of PD, and is often combined with carbidopa to reduce the dose-limiting side effects. Long-term levodopa treatment is associated with the development of motor fluctuations and peak dose dyskinesias. Dopamine Replacement Therapy (DRT) with dopamine agonists (DAs) (ropinirole and pramipexole) is used to manage complications of L-dopa treatment, however, has been associated with numerous pharmacovigilence reports. The present review attempts to narrate the multiple receptor interaction of DAs followed by the assessment of their side effects during the treatment of PD and possible remedial strategy for selective targeting of dopamine receptors to overcome these affects in therapy of Parkinson's disease. PMID:22697513

  11. Visual encoding and fixation target selection in free viewing: presaccadic brain potentials

    PubMed Central

    Nikolaev, Andrey R.; Jurica, Peter; Nakatani, Chie; Plomp, Gijs; van Leeuwen, Cees

    2013-01-01

    In scrutinizing a scene, the eyes alternate between fixations and saccades. During a fixation, two component processes can be distinguished: visual encoding and selection of the next fixation target. We aimed to distinguish the neural correlates of these processes in the electrical brain activity prior to a saccade onset. Participants viewed color photographs of natural scenes, in preparation for a change detection task. Then, for each participant and each scene we computed an image heat map, with temperature representing the duration and density of fixations. The temperature difference between the start and end points of saccades was taken as a measure of the expected task-relevance of the information concentrated in specific regions of a scene. Visual encoding was evaluated according to whether subsequent change was correctly detected. Saccades with larger temperature difference were more likely to be followed by correct detection than ones with smaller temperature differences. The amplitude of presaccadic activity over anterior brain areas was larger for correct detection than for detection failure. This difference was observed for short “scrutinizing” but not for long “explorative” saccades, suggesting that presaccadic activity reflects top-down saccade guidance. Thus, successful encoding requires local scanning of scene regions which are expected to be task-relevant. Next, we evaluated fixation target selection. Saccades “moving up” in temperature were preceded by presaccadic activity of higher amplitude than those “moving down”. This finding suggests that presaccadic activity reflects attention deployed to the following fixation location. Our findings illustrate how presaccadic activity can elucidate concurrent brain processes related to the immediate goal of planning the next saccade and the larger-scale goal of constructing a robust representation of the visual scene. PMID:23818877

  12. Cancer stem cells as targets for cancer therapy: selected cancers as examples

    PubMed Central

    Hombach-Klonisch, Sabine; Paranjothy, Ted; Wiechec, Emilia; Pocar, Paola; Mustafa, Tarek; Seifert, Anja; Zahl, Christian; Gerlach, Klaus Luis; Biermann, Katharina; Steger, Klaus; Hoang-Vu, Cuong; Schulze-Osthoff, Klaus; Los, Marek

    2010-01-01

    It is becoming increasingly evident that cancer constitutes a group of diseases involving altered stem-cell maturation/differentiation and the disturbance of regenerative processes. The observed malignant transformation is merely a symptom of normal differentiation processes gone astray rather than the primary event. This review focuses on the role of cancer stem cells (CSCs) in three common but also relatively under-investigated cancers: head and neck, ovarian, and testicular cancer. For didactic purpose, the physiology of stem cells is first introduced using hematopoietic and mesenchymal stem cells as examples. This is followed by a discussion of the (possible) role of CSCs in head and neck, ovarian, and testicular cancer. Aside from basic information about the pathophysiology of these cancers, current research results focused on the discovery of molecular markers specific to these cancers are also discussed. The last part of the review is largely dedicated to signaling pathways active within various normal and CSC types (e.g. Nanog, Nestin, Notch1, Notch2, Oct3 and 4, Wnt). Different elements of these pathways are also discussed in the context of therapeutic opportunities for the development of targeted therapies aimed at CSCs. Finally, alternative targeted anticancer therapies arising from recently identified molecules with cancer--(semi-)selective capabilities (e.g. apoptin, Brevinin-2R) are considered. PMID:18512024

  13. Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents†

    PubMed Central

    Uddin, Md. Jashim; Crews, Brenda C.; Blobaum, Anna L.; Kingsley, Philip J.; Gorden, D. Lee; McIntyre, J. Oliver; Matrisian, Lynn M.; Subbaramaiah, Kotha; Dannenberg, Andrew J.; Piston, David W.; Marnett, Lawrence J.

    2010-01-01

    Effective diagnosis of inflammation and cancer by molecular imaging is challenging because of interference from non-selective accumulation of the contrast agents in normal tissues. Here we report a series of novel fluorescence imaging agents that efficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions, and in many premalignant and malignant tumors. After either intraperitoneal or intravenous injection, these reagents become highly enriched in inflamed or tumor tissue compared to normal tissue and this accumulation provides sufficient signal for in vivo fluorescence imaging. Further, we show that only the intact parent compound is found in the region of interest. COX-2-specific delivery was unambiguously confirmed using animals bearing targeted deletions of COX-2 and by blocking the COX-2 active site with high affinity inhibitors in both in vitro and in vivo models. Because of their high specificity, contrast, and detectability, these COX-2 beacons are ideal candidates for detection of inflammatory lesions or early-stage COX-2-expressing human cancers, such as those in the esophagus, oropharynx, and colon. PMID:20430759

  14. NuMA is required for the selective induction of p53 target genes.

    PubMed

    Ohata, Hirokazu; Miyazaki, Makoto; Otomo, Ryo; Matsushima-Hibiya, Yuko; Otsubo, Chihiro; Nagase, Takahiro; Arakawa, Hirofumi; Yokota, Jun; Nakagama, Hitoshi; Taya, Yoichi; Enari, Masato

    2013-06-01

    The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription. PMID:23589328

  15. Lysosome dysfunction in the pathogenesis of kidney diseases.

    PubMed

    Surendran, Kameswaran; Vitiello, Seasson P; Pearce, David A

    2014-12-01

    The lysosome, an organelle central to macromolecule degradation and recycling, plays a pivotal role in normal cell processes, ranging from autophagy to redox regulation. Not surprisingly, lysosomes are an integral part of the renal epithelial molecular machinery that facilitates normal renal physiology. Two inherited diseases that manifest as kidney dysfunction are Fabry's disease and cystinosis, each of which is caused by a primary biochemical defect at the lysosome resulting from loss-of-function mutations in genes that encode lysosomal proteins. The functions of the lysosomes in the kidney and how lysosomal dysfunction might contribute to Fabry's disease and cystinosis are discussed. Unlike most other pediatric renal diseases, therapies are available for Fabry's disease and cystinosis, but require early diagnosis. Recent analysis of ceroid neuronal lipofuscinosis type 3 (Cln3) null mice, a mouse model of lysosomal disease that is primarily associated with neurological deficits, revealed renal functional abnormalities. As current and future therapeutics increase the life-span of those suffering from diseases like neuronal ceroid lipofuscinosis, it remains a distinct possibility that many more lysosomal disorders that primarily manifest as infant and juvenile neurodegenerative diseases may also include renal disease phenotypes. PMID:24217784

  16. Membrane Localization of ?-Amyloid 1–42 in Lysosomes

    PubMed Central

    Liu, Rui-Qin; Zhou, Qing-Hua; Ji, Shang-Rong; Zhou, Qiang; Feng, Du; Wu, Yi; Sui, Sen-Fang

    2010-01-01

    ?-Amyloid peptide (A?42) is the core protein of amyloid plaque in Alzheimer disease. The intracellular accumulation of A?42 in the endosomal/lysosomal system has been under investigation for many years, but the direct link between A?42 accumulation and dysfunction of the endosomal/lysosomal system is still largely unknown. Here, we found that both in vitro and in vivo, a major portion of A?42 was tightly inserted into and a small portion peripherally associated with the lysosomal membrane, whereas its soluble portion was minimal. We also found that the A?42 molecules inserted into the membrane tended to form multiple oligomeric aggregates, whereas A?40 peptides formed only dimers. Neutralizing lysosomal pH in differentiated PC12 cells decreased the lysosomal membrane insertion of A?42 and moderated A?42-induced lysosomal labilization and cytotoxicity. Our findings, thus, suggest that the membrane-inserted portion of A?42 accumulated in lysosomes may destabilize the lysosomal membrane and induce neurotoxicity. PMID:20430896

  17. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders

    Microsoft Academic Search

    Edward H. Schuchman; Robert J. Desnick

    2002-01-01

    The past decade has witnessed remarkable advances in our ability to treat inherited metabolic disorders, especially the lysosomal storage diseases, a group of more than 40 disorders, each of which is caused by the deficiency of a lysosomal enzyme or protein. During the past few years, both enzyme replacement and enhancement therapies have been developed to treat these disorders. This

  18. Expanding Newborn Screening for Lysosomal Disorders: Opportunities and Challenges

    ERIC Educational Resources Information Center

    Waggoner, Darrel J.; Tan, Christopher A.

    2011-01-01

    Newborn screening (NBS), since its implementation in the 1960s, has traditionally been successful in reducing mortality and disability in children with a range of different conditions. Lysosomal storage disorders (LSD) are a heterogeneous group of inherited metabolic diseases that result from lysosomal dysfunction. Based on available treatment and…

  19. Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

    PubMed

    Frenzel, Anna; Zirath, Hanna; Vita, Marina; Albihn, Ami; Henriksson, Marie Arsenian

    2011-01-01

    Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine) and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide) as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C). Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity. PMID:22132187

  20. A precisely substituted benzopyran targets androgen refractory prostate cancer cells through selective modulation of estrogen receptors.

    PubMed

    Kumar, Rajeev; Verma, Vikas; Sharma, Vikas; Jain, Ashish; Singh, Vishal; Sarswat, Amit; Maikhuri, Jagdamba P; Sharma, Vishnu L; Gupta, Gopal

    2015-03-15

    Dietary consumption of phytoestrogens like genistein has been linked with lower incidence of prostate cancer. The estradiol-like benzopyran core of genistein confers estrogen receptor-? (ER-?) selectivity that imparts weak anti-proliferative activity against prostate cancer cells. DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran (BP), a SERM designed with benzopyran core, targeted androgen independent prostate cancer (PC-3) cells 14-times more potently than genistein, ~25% more efficiently than tamoxifen and 6.5-times more actively than ICI-182780, without forfeiting significant specificity in comparison to genistein. BP increased apoptosis (annexin-V and TUNEL labeling), arrested cell cycle, and significantly increased caspase-3 activity along with mRNA expressions of estrogen receptor (ER)-? and FasL (qPCR) in PC-3 cells. In classical ERE-luc reporter assay BP behaved as a potent ER-? antagonist and ER-? agonist. Accordingly, it decreased expression of ER-? target PS2 (P<0.01) and increased expression of ER-? target TNF-? (P<0.05) genes in PC-3. ER-? deficient PC-3 (siRNA-transfected) was resistant to apoptotic and anti-proliferative actions of SERMs, including stimulation of FasL expression by BP. BP significantly inhibited phosphorylation of Akt and ERK-1/2, JNK and p38 in PC-3 (immunoblotting), and thus adopted a multi-pathway mechanism to exert a more potent anti-proliferative activity against prostate cancer cells than natural and synthetic SERMs. Its precise ER-subtype specific activity presents a unique lead structure for further optimization. PMID:25655200

  1. Targeting the XIAP/caspase-7 complex selectively kills caspase-3–deficient malignancies

    PubMed Central

    Lin, Yuan-Feng; Lai, Tsung-Ching; Chang, Chih-Kang; Chen, Chi-Long; Huang, Ming-Shyan; Yang, Chih-Jen; Liu, Hon-Ge; Dong, Jhih-Jhong; Chou, Yi-An; Teng, Kuo-Hsun; Chen, Shih-Hsun; Tian, Wei-Ting; Jan, Yi-Hua; Hsiao, Michael; Liang, Po-Huang

    2013-01-01

    Caspase-3 downregulation (CASP3/DR) in tumors frequently confers resistance to cancer therapy and is significantly correlated with a poor prognosis in cancer patients. Because CASP3/DR cancer cells rely heavily on the activity of caspase-7 (CASP7) to initiate apoptosis, inhibition of activated CASP7 (p19/p12-CASP7) by X-linked inhibitor of apoptosis protein (XIAP) is a potential mechanism by which apoptosis is prevented in those cancer cells. Here, we identify the pocket surrounding the Cys246 residue of p19/p12-CASP7 as a target for the development of a protein-protein interaction (PPI) inhibitor of the XIAP:p19/p12-CASP7 complex. Interrupting this PPI directly triggered CASP7-dependent apoptotic signaling that bypassed the activation of the apical caspases and selectively killed CASP3/DR malignancies in vitro and in vivo without adverse side effects in nontumor cells. Importantly, CASP3/DR combined with p19/p12-CASP7 accumulation correlated with the aggressive evolution of clinical malignancies and a poor prognosis in cancer patients. Moreover, targeting of this PPI effectively killed cancer cells with multidrug resistance due to microRNA let-7a-1–mediated CASP3/DR and resensitized cancer cells to chemotherapy-induced apoptosis. These findings not only provide an opportunity to treat CASP3/DR malignancies by targeting the XIAP:p19/p12-CASP7 complex, but also elucidate the molecular mechanism underlying CASP3/DR in cancers. PMID:23979166

  2. Targeting the XIAP/caspase-7 complex selectively kills caspase-3-deficient malignancies.

    PubMed

    Lin, Yuan-Feng; Lai, Tsung-Ching; Chang, Chih-Kang; Chen, Chi-Long; Huang, Ming-Shyan; Yang, Chih-Jen; Liu, Hon-Ge; Dong, Jhih-Jhong; Chou, Yi-An; Teng, Kuo-Hsun; Chen, Shih-Hsun; Tian, Wei-Ting; Jan, Yi-Hua; Hsiao, Michael; Liang, Po-Huang

    2013-09-01

    Caspase-3 downregulation (CASP3/DR) in tumors frequently confers resistance to cancer therapy and is significantly correlated with a poor prognosis in cancer patients. Because CASP3/DR cancer cells rely heavily on the activity of caspase-7 (CASP7) to initiate apoptosis, inhibition of activated CASP7 (p19/p12-CASP7) by X-linked inhibitor of apoptosis protein (XIAP) is a potential mechanism by which apoptosis is prevented in those cancer cells. Here, we identify the pocket surrounding the Cys246 residue of p19/p12-CASP7 as a target for the development of a protein-protein interaction (PPI) inhibitor of the XIAP:p19/p12-CASP7 complex. Interrupting this PPI directly triggered CASP7-dependent apoptotic signaling that bypassed the activation of the apical caspases and selectively killed CASP3/DR malignancies in vitro and in vivo without adverse side effects in nontumor cells. Importantly, CASP3/DR combined with p19/p12-CASP7 accumulation correlated with the aggressive evolution of clinical malignancies and a poor prognosis in cancer patients. Moreover, targeting of this PPI effectively killed cancer cells with multidrug resistance due to microRNA let-7a-1-mediated CASP3/DR and resensitized cancer cells to chemotherapy-induced apoptosis. These findings not only provide an opportunity to treat CASP3/DR malignancies by targeting the XIAP:p19/p12-CASP7 complex, but also elucidate the molecular mechanism underlying CASP3/DR in cancers. PMID:23979166

  3. Amino acids and mTORC1: from lysosomes to disease

    PubMed Central

    Efeyan, Alejo; Zoncu, Roberto; Sabatini, David M.

    2012-01-01

    The mechanistic target of rapamycin (mTOR) kinase controls growth and metabolism, and its deregulation underlies the pathogenesis of many diseases, including cancer, neurodegeneration, and diabetes. mTOR complex 1 (mTORC1) integrates signals arising from nutrients, energy, and growth factors, but how exactly these signals are propagated await to be fully understood. Recent findings have placed the lysosome, a key mediator of cellular catabolism, at the core of mTORC1 regulation by amino acids. A multiprotein complex that includes the Rag GTPases, Ragulator, and the v-ATPase forms an amino acid-sensing machinery on the lysosomal surface that affects the decision between cell growth and catabolism at multiple levels. The involvement of a catabolic organelle in growth signaling may have important implications for our understanding of mTORC1-related pathologies. PMID:22749019

  4. Development of targeted therapies for Parkinson's disease and related synucleinopathies.

    PubMed

    Sybertz, Edmund; Krainc, Dimitri

    2014-10-01

    Therapeutic efforts in neurodegenerative diseases have been very challenging, particularly due to a lack of validated and mechanism-based therapeutic targets and biomarkers. The basic idea underlying the novel therapeutic approaches reviewed here is that by exploring the molecular basis of neurodegeneration in a rare lysosomal disease such as Gaucher's disease (GD), new molecular targets will be identified for therapeutic development in common synucleinopathies. Accumulation of ?-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, suggesting that improved clearance of ?-synuclein may be of therapeutic benefit. To achieve this goal, it is important to identify specific mechanisms and targets involved in the clearance of ?-synuclein. Recent discovery of clinical, genetic, and pathological linkage between GD and PD offers a unique opportunity to examine lysosomal glucocerebrosidase, an enzyme mutated in GD, for development of targeted therapies in synucleinopathies. While modulation of glucocerebrosidase and glycolipid metabolism offers a viable approach to treating disorders associated with synuclein accumulation, the compounds described to date either lack the ability to penetrate the CNS or have off-target effects that may counteract or limit their capabilities to mediate the desired pharmacological action. However, recent emergence of selective inhibitors of glycosphingolipid biosynthesis and noninhibitory pharmacological chaperones of glycosphingolipid processing enzymes that gain access to the CNS provide a novel approach that may overcome some of the limitations of compounds reported to date. These new strategies may allow for development of targeted treatments for synucleinopathies that affect both children and adults. PMID:24668939

  5. Genetic Convergence of Parkinson's Disease and Lysosomal Storage Disorders.

    PubMed

    Deng, Hao; Xiu, Xiaofei; Jankovic, Joseph

    2014-08-01

    Parkinson's disease is a common progressive neurodegenerative disorder characterized by predominant degeneration of the dopaminergic neurons in the substantia nigra pars compacta and the presence of intracellular inclusions enriched in ?-synuclein, resulting in a variety motor and nonmotor symptoms. Lysosomal storage disorders are a group of disorders including Gaucher disease, Niemann-Pick disease, and neuronal ceroid lipofuscinoses caused by the defective activity of lysosomal and nonlysosomal proteins. In addition to an overlap in some clinical features between lysosomal storage disorders and Parkinson's disease, the two disorders may be also linked pathogenically. There is growing support for the notion that mutations in genes causing lysosomal storage disorders including the glucocerebrosidase gene, the sphingomyelin phosphodiesterase 1 gene, and the NPC1 gene may increase risk for developing Parkinson's disease. In this review, we discuss the recent advances in the genetic convergence of Parkinson's disease and lysosomal storage disorders, shedding new light on the understanding of shared pathogenic pathways. PMID:25099932

  6. Virotherapy targeting cyclin E overexpression in tumors with adenovirus-enhanced cancer-selective promoter.

    PubMed

    Cheng, Pei-Hsin; Rao, Xiao-Mei; Duan, Xiaoxian; Li, Xiao-Feng; Egger, Michael E; McMasters, Kelly M; Zhou, H Sam

    2015-02-01

    Oncolytic virotherapy can selectively destroy cancer cells and is a potential approach in cancer treatment. A strategy to increase tumor-specific selectivity is to control the expression of a key regulatory viral gene with a tumor-specific promoter. We have previously found that cyclin E expression is augmented in cancer cells after adenovirus (Ad) infection. Thus, the cyclin E promoter that is further activated by Ad in cancer cells may have unique properties for enhancing oncolytic viral replication. We have shown that high levels of viral E1a gene expression are achieved in cancer cells infected with Ad-cycE, in which the endogenous Ad E1a promoter was replaced with the cyclin E promoter. Ad-cycE shows markedly selective oncolytic efficacy in vitro and destroys various types of cancer cells, including those resistant to ONYX-015/dl1520. Furthermore, Ad-cycE shows a strong capacity to repress A549 xenograft tumor growth in nude mice and significantly prolongs survival. This study suggests the potential of Ad-cycE in cancer therapy and indicates the advantages of using promoters that can be upregulated by virus infection in cancer cells in development of oncolytic viruses. Key messages: Cyclin E promoter activity is high in cancer cells and enhanced by adenovirus infection. Cyclin E promoter is used to control the E1a gene of a tumor-specific oncolytic adenovirus. Ad-cycE efficiently targets cancer cells and induces oncolysis. Ad-cycE significantly repressed xenograft tumor and prolonged survival. PMID:25376708

  7. Activation of Peroxisome Proliferator-activated Receptor ? Induces Lysosomal Biogenesis in Brain Cells: IMPLICATIONS FOR LYSOSOMAL STORAGE DISORDERS.

    PubMed

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J; Sims, Katherine B; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-04-17

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) ?, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPAR?, but not PPAR? and PPAR?, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor ?, PPAR?, and PGC1? on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174

  8. ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

    SciTech Connect

    Zask, Arie; Verheijen, Jeroen C.; Curran, Kevin; Kaplan, Joshua; Richard, David J.; Nowak, Pawel; Malwitz, David J.; Brooijmans, Natasja; Bard, Joel; Svenson, Kristine; Lucas, Judy; Toral-Barza, Lourdes; Zhang, Wei-Guo; Hollander, Irwin; Gibbons, James J.; Abraham, Robert T.; Ayral-Kaloustian, Semiramis; Mansour, Tarek S.; Yu, Ker; (Wyeth)

    2009-09-18

    The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

  9. Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle

    NASA Astrophysics Data System (ADS)

    Sau, Samaresh; Agarwalla, Pritha; Mukherjee, Sudip; Bag, Indira; Sreedhar, Bojja; Pal-Bhadra, Manika; Patra, Chitta Ranjan; Banerjee, Rajkumar

    2014-05-01

    Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of `exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of `endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of `endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of `exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of `endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of `endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr00974f

  10. Network-based assessment of the selectivity of metabolic drug targets in Plasmodium falciparum with respect to human liver metabolism

    PubMed Central

    2012-01-01

    Background The search for new drug targets for antibiotics against Plasmodium falciparum, a major cause of human deaths, is a pressing scientific issue, as multiple resistance strains spread rapidly. Metabolic network-based analyses may help to identify those parasite’s essential enzymes whose homologous counterparts in the human host cells are either absent, non-essential or relatively less essential. Results Using the well-curated metabolic networks PlasmoNet of the parasite Plasmodium falciparum and HepatoNet1 of the human hepatocyte, the selectivity of 48 experimental antimalarial drug targets was analyzed. Applying in silico gene deletions, 24 of these drug targets were found to be perfectly selective, in that they were essential for the parasite but non-essential for the human cell. The selectivity of a subset of enzymes, that were essential in both models, was evaluated with the reduced fitness concept. It was, then, possible to quantify the reduction in functional fitness of the two networks under the progressive inhibition of the same enzymatic activity. Overall, this in silico analysis provided a selectivity ranking that was in line with numerous in vivo and in vitro observations. Conclusions Genome-scale models can be useful to depict and quantify the effects of enzymatic inhibitions on the impaired production of biomass components. From the perspective of a host-pathogen metabolic interaction, an estimation of the drug targets-induced consequences can be beneficial for the development of a selective anti-parasitic drug. PMID:22937810

  11. Emerging drug discovery approaches for selective targeting of “precursor” metastatic breast cancer cells: highlights and perspectives

    PubMed Central

    AAlaoui-Jamali, Moulay; Bijian, Krikor; Batist, Gerald

    2011-01-01

    Breast cancer is a prevalent disease and a major cause of morbidity and cancer-related deaths among women worldwide. A significant number of patients at the time of primary diagnosis present metastatic disease, at least to locoregional lymph nodes, which results in somewhat unpredictable prognosis that often prompts adjuvant systemic therapies of various kinds. The time course of distant recurrence is also unpredictable with some patients sustaining a recurrence within months after diagnosis, even during adjuvant treatments, while others can experience recurrence years or decades after initial diagnosis. To date, clinically approved therapeutics yielded marginal benefits for patients with systemic metastatic breast disease, since despite high clinical responses to various therapies, the patients virtually always become resistant and tumor relapses. Molecular profiling studies established that breast cancer is highly heterogeneous and encompasses diverse histological and molecular subtypes with distinct biological and clinical implications in particular in relation to the incidence of progression to metastasis. The latter has been recognized to result from late genetic events during the multistep progression proposed by the dominant theory of carcinogenesis. However, there is evidence that the dissemination of primary cancer can also be initiated at a very early stage of cancer development, originating from rare cell variants, possibly cancer stem-like cells (CSC), with invasive potential. These precursor metastatic cancer cells with stem-like properties are defined by their ability to self-renew and to regenerate cell variants, which have high plasticity and intrinsic invasive properties required for dissemination and tropism toward specific organs. Equally relevant to the CSC hypothesis for metastasis formation is the epithelial-mesenchymal transition (EMT) process, which is critical for the acquisition of cancer cell invasive behavior and for selection/gain of CSC properties. These exciting concepts have led to the formulation of various approaches for targeting precursor metastatic cells, and these have taken on greater priority in therapeutic drug discovery research by both academia and pharmaceuticals. In this review, we focus on current efforts in medicinal chemistry to develop small molecules able to target precursor metastatic cells via interference with the CSC/EMT differentiation program, self-renewal, and survival. It is not meant to be comprehensive and the reader is referred to selected reviews that provide coverage of related basic aspects. Rather, emphasis is given to promising molecules with CSC/EMT signaling at the preclinical stage and in clinical trials that are paving the way to new generations of anti-metastasis drugs. PMID:22046485

  12. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

    PubMed Central

    2012-01-01

    The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences. PMID:22697169

  13. Selected protein monitoring in histological sections by targeted MALDI-FTICR in-source decay imaging.

    PubMed

    Calligaris, David; Longuespée, Rémi; Debois, Delphine; Asakawa, Daiki; Turtoi, Andrei; Castronovo, Vincent; Noël, Agnès; Bertrand, Virginie; De Pauw-Gillet, Marie-Claire; De Pauw, Edwin

    2013-02-19

    Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of spectral pattern to define regions of interest. However, the identification and the differential quantitative analysis of proteins require off line or in situ proteomic methods using enzymatic digestion. The rapid identification of biomarkers holds great promise for diagnostic research, but the major obstacle is the absence of a rapid and direct method to detect and identify with a sufficient dynamic range a set of specific biomarkers. In the current work, we present a proof of concept for a method allowing one to identify simultaneously a set of selected biomarkers on histological slices with minimal sample treatment using in-source decay (ISD) MSI and MALDI-Fourier transform ion cyclotron resonance (FTICR). In the proposed method, known biomarkers are spotted next to the tissue of interest, the whole MALDI plate being coated with 1,5-diaminonaphthalene (1,5-DAN) matrix. The latter enhances MALDI radical-induced ISD, providing large tags of the amino acid sequences. Comparative analysis of ISD fragments between the reference spots and the specimen in imaging mode allows for unambiguous identification of the selected biomarker while preserving full spatial resolution. Moreover, the high resolution/high mass accuracy provided by FTICR mass spectrometry allows the identification of proteins. Well-resolved peaks and precise measurements of masses and mass differences allow the construction of reliable sequence tags for protein identification. The method will allow the use of MALDI-FTICR MSI as a method for rapid targeted biomarker detection in complement to classical histology. PMID:23323725

  14. Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1.

    PubMed

    Nuttall, Stewart D; Humberstone, Karen S; Krishnan, Usha V; Carmichael, Jennifer A; Doughty, Larissa; Hattarki, Meghan; Coley, Andrew M; Casey, Joanne L; Anders, Robin F; Foley, Michael; Irving, Robert A; Hudson, Peter J

    2004-04-01

    The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications. PMID:14997552

  15. Selective Isolation and Ansamycin-Targeted Screenings of Commensal Actinomycetes from the “Maytansinoids-Producing” Arboreal Trewia nudiflora

    Microsoft Academic Search

    Na Zhu; Peiji Zhao; Yuemao Shen

    2009-01-01

    To verify the hypothesis on the involvement of commensal actinomycetes in the biosynthesis of plant maytansinoids that belong\\u000a to the ansamycin family, selective isolation and targeted screenings were conducted. In total, 164 endophytic actinomycetes\\u000a isolates were obtained from the roots, twigs, seeds, callus, and germ-free seedlings of “maytansinoids-producing” Trewia nudiflora Linn. by selective isolation methods. Crude extracts of the isolates

  16. Microfluidic enrichment of a target cell type from a heterogenous suspension by adhesion-based negative selection.

    PubMed

    Green, James V; Murthy, Shashi K

    2009-08-01

    The enrichment or isolation of a selected type of cells in a heterogeneous suspension is challenging when the surface markers of these cells are not completely known. Here, we present a 3-stage arrangement of peptide-coated microfluidic channels that can recover a small number of adipose-derived stem cells (ADSCs) from a heterogenous suspension by negative selection depletion of the non-target cell types. PMID:19606304

  17. Identifying modulators of CXC receptors 3 and 4 with tailored selectivity using multi-target docking.

    PubMed

    Schmidt, Denis; Bernat, Viachaslau; Brox, Regine; Tschammer, Nuska; Kolb, Peter

    2015-03-20

    The G protein-coupled receptors of the C-X-C subfamily form a group among the chemokine receptors whose endogenous ligands are peptides with a common Cys-X-Cys motif. The CXC chemokine receptors 3 and 4 (CXCR3, CXCR4), which are investigated in this study, are linked to severe diseases such as cancer, multiple sclerosis, and HIV infections. Of particular interest, this receptor pair potentially forms a target for a polypharmacological drug treatment. Considering known ligands from public databases, such dual binders have not been identified yet. We therefore applied large-scale docking to the structure of CXCR4 and a homology model of CXCR3 with the goal to predict such dual binders, as well as compounds selective for either one of the receptors. Using signaling and biochemical assays, we showed that more than 50% of these predictions were correct in each category, yielding ligands with excellent binding efficiencies. These results highlight that docking is a suitable tool for the identification of ligands with tailored binding profiles to GPCRs, even when using homology models. More importantly, we present novel CXCR3-CXCR4 dual modulators that might pave the road to understanding the mechanisms of polypharmacological inhibition of these receptors. PMID:25398025

  18. Folic Acid-conjugated europium complexes as luminescent probes for selective targeting of cancer cells.

    PubMed

    Quici, Silvio; Casoni, Alessandro; Foschi, Francesca; Armelao, Lidia; Bottaro, Gregorio; Seraglia, Roberta; Bolzati, Cristina; Salvarese, Nicola; Carpanese, Debora; Rosato, Antonio

    2015-02-26

    We report the synthesis of three optical probes (Eu(3+)?1, Eu(3+)?2, and Eu(3+)?3) having a luminescent Eu complex (signaling unit) bonded in different positions to folic acid (FA), the folate receptor (FR) targeting unit. The structures of the two regioisomers Eu(3+)?1 and Eu(3+)?2 were assigned by mass spectrometric experiments. The optical properties and stability of these probes were assessed in phosphate-buffered saline, cell culture medium, rat serum, and cellular lysate, and results indicated that they are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells having high (SKOV-3), intermediate (OVCAR-3), low (IGROV-1), or null (A2780) expression of FRs. The internalized probe, evaluated in SKOV-3, IGROV-1, and A2780 cells, was in the order Eu(3+)?2 > Eu(3+)?1 > Eu(3+)?3. No internalization was observed for A2780 cells. Such results, together with those obtained in competition experiments of FA versus Eu(3+)?2 and FA or Eu(3+)?2 versus (3)H-FA, indicate that internalization is receptor-mediated and that Eu(3+)?2 shows high selectivity and specificity for FR. PMID:25602505

  19. PFI-1 – A highly Selective Protein Interaction Inhibitor Targeting BET Bromodomains

    PubMed Central

    Picaud, Sarah; Costa, David Da; Thanasopoulou, Angeliki; Filippakopoulos, Panagis; Fish, Paul V.; Philpott, Martin; Fedorov, Oleg; Brennan, Paul; Bunnage, Mark E.; Owen, Dafydd R.; Bradner, James E.; Taniere, Philippe; O’Sullivan, Brendan; Müller, Susanne; Schwaller, Juerg; Stankovic, Tatjana; Knapp, Stefan

    2013-01-01

    Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and anti-apoptotic genes as well as for cell cycle progression. BET proteins are recruited to transcriptionally active chromatin via their two N-terminal bromodomains (BRDs), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional down-regulation of a number of oncogenes providing a novel pharmacological strategy for the treatment of cancer. Here we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1 that efficiently blocks the interaction of BET BRDs with acetylated histone tails. Co-crystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukaemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell cycle arrest, down-regulation of MYC expression as well as induction of apoptosis and induces differentiation of primary leukaemic blasts. Intriguingly, cells exposed to PFI-1 showed significant down-regulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10 providing an alternative strategy for the specific inhibition of this well established oncology target. PMID:23576556

  20. Intracellular selection of peptide inhibitors that target disulphide-bridged A?42 oligomers.

    PubMed

    Acerra, Nicola; Kad, Neil M; Cheruvara, Harish; Mason, Jody M

    2014-09-01

    The ?-amyloid (A?) peptide aggregates into a number of soluble and insoluble forms, with soluble oligomers thought to be the primary factor implicated in Alzheimer's disease pathology. As a result, a wide range of potential aggregation inhibitors have been developed. However, in addition to problems with solubility and protease susceptibility, many have inadvertently raised the concentration of these soluble neurotoxic species. Sandberg et al. previously reported a ?-hairpin stabilized variant of A?42 that results from an intramolecular disulphide bridge (A21C/A31C; A?42cc), which generates highly toxic oligomeric species incapable of converting into mature fibrils. Using an intracellular protein-fragment complementation (PCA) approach, we have screened peptide libraries using E. coli that harbor an oxidizing environment to permit cytoplasmic disulphide bond formation. Peptides designed to target either the first or second ?-strand have been demonstrated to bind to A?42cc, lower amyloid cytotoxicity, and confer bacterial cell survival. Peptides have consequently been tested using wild-type A?42 via ThT binding assays, circular dichroism, MTT cytotoxicity assays, fluorescence microscopy, and atomic force microscopy. Results demonstrate that amyloid-PCA selected peptides function by both removing amyloid oligomers as well as inhibiting their formation. These data further support the use of semirational design combined with intracellular PCA methodology to develop A? antagonists as candidates for modification into drugs capable of slowing or even preventing the onset of AD. PMID:24947815

  1. A selective mitochondrial-targeted chlorambucil with remarkable cytotoxicity in breast and pancreatic cancers.

    PubMed

    Millard, Melissa; Gallagher, John D; Olenyuk, Bogdan Z; Neamati, Nouri

    2013-11-27

    Nitrogen mustards, widely used as chemotherapeutics, have limited safety and efficacy. Mitochondria lack a functional nucleotide excision repair mechanism to repair DNA adducts and are sensitive to alkylating agents. Importantly, cancer cells have higher intrinsic mitochondrial membrane potential (??mt) than normal cells. Therefore, selectively targeting nitrogen mustards to cancer cell mitochondria based on ??mt could overcome those limitations. Herein, we describe the design, synthesis, and evaluation of Mito-Chlor, a triphenylphosphonium derivative of the nitrogen mustard chlorambucil. We show that Mito-Chlor localizes to cancer cell mitochondria where it acts on mtDNA to arrest cell cycle and induce cell death, resulting in a 80-fold enhancement of cell kill in a panel of breast and pancreatic cancer cell lines that are insensitive to the parent drug. Significantly, Mito-Chlor delayed tumor progression in a mouse xenograft model of human pancreatic cancer. This is a first example of repurposing chlorambucil, a drug not used in breast and pancreatic cancer treatment, as a novel drug candidate for these diseases. PMID:24147900

  2. SIMPLE/LITAF expression induces the translocation of the ubiquitin ligase itch towards the lysosomal compartments.

    PubMed

    Eaton, Heather E; Desrochers, Guillaume; Drory, Samuel B; Metcalf, Julie; Angers, Annie; Brunetti, Craig R

    2011-01-01

    LITAF is a small cellular protein with an unknown function. The C-terminus of LITAF contains a highly conserved domain termed the SIMPLE-like domain (SLD), while the N-terminus contains two PPXY motifs that mediate protein-protein interactions with WW-domain containing proteins. LITAF also harbors two endosome/lysosome targeting sequences at its C-terminus, but there has been conflicting reports regarding its intracellular localization. Here, we demonstrate that LITAF is localized to the late endosome/lysosomal compartment in a variety of cell lines. We also show that Itch, a WW-domain containing protein, and LITAF strongly interact and that this interaction depends on the two PPXY motifs in the N-terminus of LITAF. Interestingly, co-expression of LITAF with Itch induces major changes in Itch intracellular localization, bringing Itch from the trans-Golgi network to lysosomes. We show that this re-localization is dependent upon the interaction with the PPXY sequences of LITAF, since disruption of these binding motifs completely abrogates Itch re-localization. PMID:21326863

  3. Lysosomal Trafficking of TGFBIp via Caveolae-Mediated Endocytosis

    PubMed Central

    Choi, Seung-il; Maeng, Yong-Sun; Kim, Tae-im; Lee, Yangsin; Kim, Yong-Sun; Kim, Eung Kweon

    2015-01-01

    Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin ?V?3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy. PMID:25853243

  4. C. elegans BLOC-1 Functions in Trafficking to Lysosome-Related Gut Granules

    PubMed Central

    Hermann, Greg J.; Scavarda, Emily; Weis, Allison M.; Saxton, Daniel S.; Thomas, Laura L.; Salesky, Rebecca; Somhegyi, Hannah; Curtin, Thomas P.; Barrett, Alec; Foster, Olivia K.; Vine, Annalise; Erlich, Katherine; Kwan, Elizabeth; Rabbitts, Beverley M.; Warren, Kaila

    2012-01-01

    The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(?) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(?) and BLOC-1(?) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans. PMID:22916203

  5. MDMA induces cardiac contractile dysfunction through autophagy upregulation and lysosome destabilization in rats.

    PubMed

    Shintani-ishida, Kaori; Saka, Kanju; Yamaguchi, Koji; Hayashida, Makiko; Nagai, Hisashi; Takemura, Genzou; Yoshida, Ken-ichi

    2014-05-01

    The underlying mechanisms of cardiotoxicity of 3,4-methylenedioxymethylamphetamine (MDMA, "ecstasy") abuse are unclear. Autophagy exerts either adaptive or maladaptive effects on cardiac function in various pathological settings, but nothing is known on the role of autophagy in the MDMA cardiotoxicity. Here, we investigated the mechanism through which autophagy may be involved in MDMA-induced cardiac contractile dysfunction. Rats were injected intraperitoneally with MDMA (20mg/kg) or saline. Left ventricular (LV) echocardiography and LV pressure measurement demonstrated reduction of LV systolic contractility 24h after MDMA administration. Western blot analysis showed a time-dependent increase in the levels of microtubule-associated protein light chain 3-II (LC3-II) and cathepsin-D after MDMA administration. Electron microscopy showed the presence of autophagic vacuoles in cardiomyocytes. MDMA upregulated phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) at Thr172, mammalian target of rapamycin (mTOR) at Thr2446, Raptor at Ser792, and Unc51-like kinase (ULK1) at Ser555, suggesting activation of autophagy through the AMPK-mTOR pathway. The effects of autophagic inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) on LC3-II levels indicated that MDMA enhanced autophagosome formation, but attenuated autophagosome clearance. MDMA also induced release of cathepsins into cytosol, and western blotting and electron microscopy showed cardiac troponin I (cTnI) degradation and myofibril damage, respectively. 3-MA, CQ, and a lysosomal inhibitor, E64c, inhibited cTnI proteolysis and improved contractile dysfunction after MDMA administration. In conclusion, MDMA causes lysosome destabilization following activation of the autophagy-lysosomal pathway, through which released lysosomal proteases damage myofibrils and induce LV systolic dysfunction in rat heart. PMID:24491919

  6. Cystine Transport is Defective in Isolated Leukocyte Lysosomes from Patients with Cystinosis

    Microsoft Academic Search

    W. A. Gahl; N. Bashan; F. Tietze; I. Bernardini; J. D. Schulman

    1982-01-01

    The activity of a cystine transport system in lysosomes prepared from the leukocytes of patients with cystinosis was found to be deficient. In normal subjects, this system was resistant to N-ethylmaleimide and demonstrated saturation kinetics. Lysosomes from individuals heterozygous for cystinosis demonstrated a reduced maximum velocity for cystine egress from lysosomes. The rate of cystine escape from normal lysosomes was

  7. Lysosomal Impairment in Parkinson's Disease Benjamin Dehay, PhD,1

    E-print Network

    Caldwell, Guy

    in lysosomal- related genes, such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2Lysosomal Impairment in Parkinson's Disease Benjamin Dehay, PhD,1 * Marta Martinez-Vicente, PhD,2. In addition, PD-linked mutations and post- translational modifications of a-synuclein impair its own lysosomal

  8. Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

    Microsoft Academic Search

    Johann Soret; Nadia Bakkour; Sophie Maire; Sébastien Durand; Latifa Zekri; Mathieu Gabut; Weronika Fic; Gilles Divita; Christian Rivalle; Daniel Dauzonne; Chi Hung Nguyen; Philippe Jeanteur; Jamal Tazi

    2005-01-01

    The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction.

  9. Oncolytic virotherapy: Molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses

    Microsoft Academic Search

    Z. Sheng Guo; Stephen H. Thorne; David L. Bartlett

    2008-01-01

    Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induce cancer cells

  10. Efficacy Trial of a Selective Prevention Program Targeting Both Eating Disorder Symptoms and Unhealthy Weight Gain among Female College Students

    ERIC Educational Resources Information Center

    Stice, Eric; Rohde, Paul; Shaw, Heather; Marti, C. Nathan

    2012-01-01

    Objective: Evaluate a selective prevention program targeting both eating disorder symptoms and unhealthy weight gain in young women. Method: Female college students at high-risk for these outcomes by virtue of body image concerns (N = 398; M age = 18.4 years, SD = 0.6) were randomized to the Healthy Weight group-based 4-hr prevention program,…

  11. Abstract--Our sensor selection algorithm targets the problem of global self-localization of multi-sensor mobile

    E-print Network

    Koschan, Andreas

    Abstract--Our sensor selection algorithm targets the problem of global self-localization of multi-sensor mobile robots. The algorithm builds on the probabilistic reasoning using Bayes filters to estimate sensor measurement uncertainty and sensor validity in robot localization. For quantifying measurement uncertainty we

  12. Effects of Interior Bezels of Tiled-Monitor Large Displays on Visual Search, Tunnel Steering, and Target Selection

    E-print Network

    Balakrishnan, Ravin

    in various application domains. However, how their interior bezels affect user performance and behavior has-tunnel steering, but not to target selection. In addition, we discuss how inte- rior bezels affect user behaviors understand bezel effects on user performances and behaviors in visual search, straight tunnel steering

  13. Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

    Microsoft Academic Search

    Rafael P. Vázquez-Manrique; James C. Legg; Birgitta Olofsson; Sung Ly; Howard A. Baylis

    2010-01-01

    Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans,

  14. Deciding Where to Attend: Priming of Pop-Out Drives Target Selection

    ERIC Educational Resources Information Center

    Brascamp, Jan W.; Blake, Randolph; Kristjansson, Arni

    2011-01-01

    With attention and eye-movements humans orient to targets of interest. This orienting occurs faster when the same target repeats: priming of pop-out (PoP). While reaction times (RTs) can be important, PoP's real function could be to steer "where" to orient, a possibility underexposed in many current paradigms, as these predesignate a target to…

  15. In Vitro HIV-1 Selective Integration into the Target Sequence and Decoy-Effect of the Modified Sequence

    E-print Network

    Tatsuaki Tsuruyama; Tonau Nakai; Takuya Hiratsuka; Guang Jin; Takuro Nakamura

    Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 59-(GTCCCTTCCCAGT) n(ACTGGGAAGGGAC) n-39, and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and

  16. Acidic nanoparticles are trafficked to lysosomes and restore an acidic lysosomal pH and degradative function to compromised ARPE-19 cells.

    PubMed

    Baltazar, Gabriel C; Guha, Sonia; Lu, Wennan; Lim, Jason; Boesze-Battaglia, Kathleen; Laties, Alan M; Tyagi, Puneet; Kompella, Uday B; Mitchell, Claire H

    2012-01-01

    Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity. PMID:23272048

  17. Cholesterol Transport through Lysosome-Peroxisome Membrane Contacts.

    PubMed

    Chu, Bei-Bei; Liao, Ya-Cheng; Qi, Wei; Xie, Chang; Du, Ximing; Wang, Jiang; Yang, Hongyuan; Miao, Hong-Hua; Li, Bo-Liang; Song, Bao-Liang

    2015-04-01

    Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases. PMID:25860611

  18. Genetic Regulation of Caenorhabditis elegans Lysosome Related Organelle Function

    E-print Network

    Soukas, Alexander A.

    Lysosomes are membrane-bound organelles that contain acid hydrolases that degrade cellular proteins, lipids, nucleic acids, and oligosaccharides, and are important for cellular maintenance and protection against age-related ...

  19. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  20. Aging: central role for autophagy and the lysosomal degradative system.

    PubMed

    Rajawat, Yogendra S; Hilioti, Zoe; Bossis, Ioannis

    2009-07-01

    The lysosomal network is the major intracellular proteolytic system accounting for more than 98% of long-lived bulk protein degradation and recycling particularly in tissues such as liver and muscles. Lysosomes are the final destination of intracellular damaged structures, identified and sequestered by the processes of macroautophagy and chaperone-mediated autophagy (CMA). In the process of macroautophagy, long-lived proteins and other macromolecular aggregates and damaged intracellular organelles are first engulfed by autophagosomes. Autophagosomes themselves have limited degrading capacity and rely on fusion with lysosomes. Unlike macroautophagy, CMA does not require intermediate vesicle formation and the cytosolic proteins recognized by this pathway are directly translocated to the lysosomal membrane. Aging is a universal phenomenon characterized by progressive deterioration of cells and organs due to accumulation of macromolecular and organelle damage. The continuous removal of worn-out components and replacement with newly synthesized ones ensures cellular homeostasis and delays the aging process. Growing evidence indicate that the rate of autophagosome formation and maturation and the efficiency of autophagosome/lysosome fusion decline with age. In addition, a progressive increase in intralysosomal concentration of free radicals and the age pigment lipofuscin further diminish the efficiency of lysosomal protein degradation. Therefore, integrity of the autophagosomal-lysosomal network appears to be critical in the progression of aging. Discovery of the genes involved in the process of autophagy has provided insight into the various molecular pathways that may be involved in aging and senescence. In this review, we discuss the cellular and molecular mechanisms involved in autophagy and the role of autophagosome/lysosome network in the aging process. PMID:19427410

  1. Influence of Membrane Physical State on the Lysosomal Proton Permeability

    Microsoft Academic Search

    G.-J. Zhang; H.-W. Liu; L. Yang; Y.-G. Zhong; Y.-Z. Zheng

    2000-01-01

    .   Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane\\u000a potential with 3,3?-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.\\u000a Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol

  2. From Target Selection to Post-Stimulation Analysis: Example of an Unconventional Faulted Reservoir

    NASA Astrophysics Data System (ADS)

    LeCalvez, J. H.; Williams, M.; Xu, W.; Stokes, J.; Moros, H.; Maxwell, S. C.; Conners, S.

    2011-12-01

    As the global balance of supply and demand forces the hydrocarbon industry toward unconventional resources, technology- and economics-driven shale oil and gas production is gaining momentum throughout many basins worldwide. Production from such unconventional plays is facilitated by massive hydraulic fracturing treatments aimed at increasing permeability and reactivating natural fractures. Large-scale faulting and fracturing partly control stress distribution, hence stimulation-derived hydraulically-induced fracture systems development. Therefore, careful integrated approaches to target selection, treatment staging, and stimulation methods need to be used to economically maximize ultimate hydrocarbon recovery. We present a case study of a multistage, multilateral stimulation project in the Fort Worth Basin, Texas. Wells had to be drilled within city limits in a commercially developing building area. Well locations and trajectories were determined in and around large-scale faults using 3D surface seismic with throws varying from seven to thirty meters. As a result, three horizontal wells were drilled in the Lower Barnett Shale section, 150 m apart with the central well landed about 25 m shallower than the outside laterals. Surface seismic indicates that the surface locations are on top of a major fault complex with the lateral sections drilling away from the major fault system and through a smaller fault. Modeling of the borehole-based microseismic monitoring options led to the selection of an optimum set of configurations given the operational restrictions faced: monitoring would mainly take place using a horizontal array to be tractored downhole and moved according to the well and stage to be monitored. Wells were completed using a perf-and-plug approach allowing for each stimulation stage to obtain a precise orientation of the various three-component accelerometers of the monitoring array as well as the calibration of the velocity model used to process the microseismic data acquired. Real-time microseismic monitoring allowed (i) to avoid the water-bearing formation below the zone of interest, (ii) to bypass the faulted zone, and (iii) to modify as needed the perforation and stimulation plans. Completion led to an initial gas production of over 3 MMCF/day each. Early decline rates confirm successful completion in avoiding the faulted areas. Initial observations of the slickwater fracturing stimulation treatments for these three wells using an integrated approach involving mechanical modelling calibrated using microseismic data indicate that (i) a long bi-wing-like fracture system initiated prior to being followed by a complex fracture network; thus, explaining the fact that some events are mapped relatively far away from the injection site, (ii) proppant generally settled down in the near wellbore area during the fracture network development due to rapid decrease of fluid flow velocity away from the injection side. Initial b-value results seem to indicate that the target reservoir is naturally fractured and that the influence of a large fault system in the vicinity of the treated zone could be asserted.

  3. HELIOSEISMOLOGY OF PRE-EMERGING ACTIVE REGIONS. I. OVERVIEW, DATA, AND TARGET SELECTION CRITERIA

    SciTech Connect

    Leka, K. D.; Barnes, G.; Birch, A. C.; Dunn, T.; Javornik, B.; Braun, D. C. [NorthWest Research Associates, Boulder, CO 80301 (United States)] [NorthWest Research Associates, Boulder, CO 80301 (United States); Gonzalez-Hernandez, I. [National Solar Observatory, Tucson, AZ 85719 (United States)] [National Solar Observatory, Tucson, AZ 85719 (United States)

    2013-01-10

    This first paper in a series describes the design of a study testing whether pre-appearance signatures of solar magnetic active regions were detectable using various tools of local helioseismology. The ultimate goal is to understand flux-emergence mechanisms by setting observational constraints on pre-appearance subsurface changes, for comparison with results from simulation efforts. This first paper provides details of the data selection and preparation of the samples, each containing over 100 members, of two populations: regions on the Sun that produced a numbered NOAA active region, and a 'control' sample of areas that did not. The seismology is performed on data from the GONG network; accompanying magnetic data from SOHO/MDI are used for co-temporal analysis of the surface magnetic field. Samples are drawn from 2001-2007, and each target is analyzed for 27.7 hr prior to an objectively determined time of emergence. The results of two analysis approaches are published separately: one based on averages of the seismology- and magnetic-derived signals over the samples, another based on Discriminant Analysis of these signals, for a statistical test of detectable differences between the two populations. We include here descriptions of a new potential-field calculation approach and the algorithm for matching sample distributions over multiple variables. We describe known sources of bias and the approaches used to mitigate them. We also describe unexpected bias sources uncovered during the course of the study and include a discussion of refinements that should be included in future work on this topic.

  4. Therapeutic approaches for lysosomal storage diseases

    PubMed Central

    Pastores, Gregory M.

    2010-01-01

    The lysosomal storage disorders (LSDs) comprise a heterogeneous group of inborn errors of metabolism characterized by tissue substrate deposits, most often caused by a deficiency of the enzyme normally responsible for catabolism of various byproducts of cellular turnover. Individual entities are typified by involvement of multiple body organs, in a pattern reflecting the sites of substrate storage. It is increasingly recognized that one or more processes, such as aberrant inflammation, dysregulation of apoptosis and/or defects of autophagy, may mediate organ dysfunction or failure. Several therapeutic options for various LSDs have been introduced, including hematopoietic stem cell transplantation, enzyme replacement therapy and substrate reduction therapy. Additional strategies are being explored, including the use of pharmacological chaperones and gene therapy. Most LSDs include a variant characterized by primary central nervous system (CNS) involvement. At present, therapy of the CNS manifestations remains a major challenge because of the inability to deliver therapeutic agents across the intact blood—brain barrier. With improved understanding of underlying disease mechanisms, additional therapeutic options may be developed, complemented by various strategies to deliver the therapeutic agent(s) to recalcitrant sites of pathology such as brain, bones and lungs. PMID:23148162

  5. Clinical studies in lysosomal storage diseases

    PubMed Central

    Boudes, Pol F

    2013-01-01

    Lysosomal storage disorders (LSDs) consist of over 40 diseases, some of which are amenable to treatment. In this review, we consider the regulatory context in which LSDs studies are performed, highlight design specificities and explore operational challenges. Orphan drug legislations, both in Europe and US, were effective to stimulate LSDs drug development. However, regulators flexibilities toward approval vary leading to global discrepancies in access to treatments. Study designs are constrained because few patients can be studied. This implies LSDs treatments need to demonstrate large levels of clinical efficacy. If not, an appropriate level of evidence is difficult to achieve. While biomarkers could address this issue, none have been truly accepted as primary outcome. Enrichment of study population can increase the chance of success, especially with clinical outcomes. Adaptive designs are operationally challenging. Innovative methods of analysis can be used, notably using a patient as his/her own control and responder analysis. The use of extension phases and patient registries as a source of historical comparison can facilitate data interpretation. Operationally, few patients are available per centers and multiple centers need to be initiated in multiple countries. This impacts time-lines and budget. In the future, regulators flexibility will be essential to provide patients access to innovative treatments. PMID:25003011

  6. [Enzyme replacement therapy of lysosomal storage diseases].

    PubMed

    Germain, D P; Boucly, C; Carlier, R Y; Caudron, E; Charlier, P; Colas, F; Jabbour, F; Martinez, V; Mokhtari, S; Orlikowski, D; Pellegrini, N; Perronne, C; Prigent, H; Rubinsztajn, R; Benistan, K

    2010-12-01

    Extraction and purification of an acid ?-glucosidase from human placenta (alglucerase) for the treatment of Gaucher disease, replaced a few years later by a recombinant enzyme (imiglucérase, Cerezyme(®)), has paved the way to the development of enzyme replacement therapies (ERT) for the treatment of lysosomal storage diseases (LSD) among which Fabry disease for which the long-term efficacy of the two currently available preparations (agalsidase alfa, Replagal(®) and Fabrazyme(®)) is still being investigated. Mucopolysaccharidosis (MPS) type I (Hurler and Scheie diseases), II (Hunter syndrome) and VI (Maroteaux-Lamy disease) also benefit from ERT using laronidase (Aldurazyme(®)), idursulfase (Elaprase(®)) and galsulfase (Naglazyme(®)), respectively. ERT reduces the hepatosplenomegaly and improves the physical and respiratory capacities of MPS patients with a globally acceptable safety profile although the possibility of infusion-associated should always be kept in mind. Alglucosidase alpha (Myozyme(®)) improves the cardiomyopathy and life expectancy of infants suffering from Pompe disease and is under evaluation for the treatment of the juvenile and adult forms of the disease. CNS involvement remains a major challenge for many LSD and innovative research and approaches are needed to address the fact that recombinant enzymes do not cross the blood-brain barrier and therefore are not expected to lead to any improvement in CNS damages, except if alternative routes such as intrathecal administration would be developed. Molecular chaperones (e.g. migalastat for Fabry disease) and inhibitors of glucosylceramide synthesis (e.g. eliglustat tartrate for Gaucher disease) are currently under investigation in various clinical trials. PMID:21211680

  7. Caging of plumbagin on silver nanoparticles imparts selectivity and sensitivity to plumbagin for targeted cancer cell apoptosis.

    PubMed

    Duraipandy, N; Lakra, Rachita; Kunnavakkam Vinjimur, Srivatsan; Samanta, Debasis; K, Purna Sai; Kiran, Manikantan Syamala

    2014-11-01

    Plumbagin is a nutraceutical with potent anti-cancer activity. However, the therapeutic efficacy of plumbagin is overshadowed by the lack of sensitivity and selectivity towards cancer cells. The present study evaluated the use of nano-biotechnological intervention to cage plumbagin in silver nanoparticles for selective targeting of its biological effects towards cancerous cells. Caging of plumbagin in silver nanoparticles imparted selectivity and sensitivity to plumbagin for selective killing of cancer cells by altering the redox signalling events in the cancer cells. The selectivity and sensitivity of plumbagin towards cancer cells was due to the cumulative expression of the properties of plumbagin and nanoparticles which specifically affected the differential cancer cell microenvironment by altering the pyruvate kinase activity that regulates the ROS challenge in cancerous cells. The positive surface charge of plumbagin caged silver nanoparticles (PCSN) aids in getting them targeted towards anionic cancerous cells due to the exposed terminal carboxyl group of sialic acid residues. Furthermore, we observed that the effective concentration of the drug required to induce apoptosis was brought down to 50% upon caging of plumbagin on silver nanoparticles. We observed no such effect with the individual compound alone. The results indicated that the physico-chemical and biochemical properties of plumbagin significantly changed after conjugation with nanomaterials that facilitated "adding-in" therapeutical values to plumbagin which would otherwise be overshadowed by its lack of sensitivity and selectivity towards cancer cells. PMID:25188862

  8. Comparing the Selection and Placement of Best Management Practices in Improving Water Quality Using a Multiobjective Optimization and Targeting Method

    PubMed Central

    Chiang, Li-Chi; Chaubey, Indrajeet; Maringanti, Chetan; Huang, Tao

    2014-01-01

    Suites of Best Management Practices (BMPs) are usually selected to be economically and environmentally efficient in reducing nonpoint source (NPS) pollutants from agricultural areas in a watershed. The objective of this research was to compare the selection and placement of BMPs in a pasture-dominated watershed using multiobjective optimization and targeting methods. Two objective functions were used in the optimization process, which minimize pollutant losses and the BMP placement areas. The optimization tool was an integration of a multi-objective genetic algorithm (GA) and a watershed model (Soil and Water Assessment Tool—SWAT). For the targeting method, an optimum BMP option was implemented in critical areas in the watershed that contribute the greatest pollutant losses. A total of 171 BMP combinations, which consist of grazing management, vegetated filter strips (VFS), and poultry litter applications were considered. The results showed that the optimization is less effective when vegetated filter strips (VFS) are not considered, and it requires much longer computation times than the targeting method to search for optimum BMPs. Although the targeting method is effective in selecting and placing an optimum BMP, larger areas are needed for BMP implementation to achieve the same pollutant reductions as the optimization method. PMID:24619160

  9. In Vitro HIV-1 Selective Integration into the Target Sequence and Decoy-Effect of the Modified Sequence

    PubMed Central

    Tsuruyama, Tatsuaki; Nakai, Tonau; Hiratsuka, Takuya; Jin, Guang; Nakamura, Takuro; Yoshikawa, Kenichi

    2010-01-01

    Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5?-(GTCCCTTCCCAGT)n(ACTGGGAAGGGAC)n-3?, and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA. PMID:21079805

  10. Identification of target-binding peptide motifs by high-throughput sequencing of phage-selected peptides.

    PubMed

    Rentero Rebollo, Inmaculada; Sabisz, Michal; Baeriswyl, Vanessa; Heinis, Christian

    2014-12-16

    High-throughput sequencing was previously applied to phage-selected peptides in order to gain insight into the abundance and diversity of isolated peptides. Herein we developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides for the purpose of identifying target-binding peptide motifs. We applied the procedure to analyze bicyclic peptides isolated against five different protein targets: sortase A, urokinase-type plasminogen activator, coagulation factor XII, plasma kallikrein and streptavidin. We optimized sequence data filters to reduce biases originating from the sequencing method and developed sequence correction algorithms to prevent identification of false consensus motifs. With our strategy, we were able to identify rare target-binding peptide motifs, as well as to define more precisely consensus sequences and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and it facilitates identification of epitopes. We furthermore show that binding motifs can be identified after a single round of phage selection. Such a selection regimen reduces propagation-related bias and may facilitate application of phage display in non-specialized laboratories, as procedures such as bacterial infection, phage propagation and purification are not required. PMID:25348396

  11. Identification of target-binding peptide motifs by high-throughput sequencing of phage-selected peptides

    PubMed Central

    Rentero Rebollo, Inmaculada; Sabisz, Michal; Baeriswyl, Vanessa; Heinis, Christian

    2014-01-01

    High-throughput sequencing was previously applied to phage-selected peptides in order to gain insight into the abundance and diversity of isolated peptides. Herein we developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides for the purpose of identifying target-binding peptide motifs. We applied the procedure to analyze bicyclic peptides isolated against five different protein targets: sortase A, urokinase-type plasminogen activator, coagulation factor XII, plasma kallikrein and streptavidin. We optimized sequence data filters to reduce biases originating from the sequencing method and developed sequence correction algorithms to prevent identification of false consensus motifs. With our strategy, we were able to identify rare target-binding peptide motifs, as well as to define more precisely consensus sequences and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and it facilitates identification of epitopes. We furthermore show that binding motifs can be identified after a single round of phage selection. Such a selection regimen reduces propagation-related bias and may facilitate application of phage display in non-specialized laboratories, as procedures such as bacterial infection, phage propagation and purification are not required. PMID:25348396

  12. Preparation of four fluorine-18-labeled estrogens and their selective uptakes in target tissues of immature rats

    SciTech Connect

    Kiesewetter, D.O.; Kilbourn, M.R.; Landvatter, S.W.; Heiman, D.F.; Katzenellenbogen, J.A.; Welch, M.J.

    1984-11-01

    Four fluorine-18-labeled estrogens have been prepared by simple displacement reactions utilizing reactive trifluoromethane sulfonate (triflate) precursors and F-18 fluoride ion. All four fluoroestrogens have high affinity for the estrogen receptor. In immature female rats, they are taken up by target tissues, such as the uterus, with very high selectivity: uterus-to-blood ratios at 1 hr are: Compound 1, 39; Compound 2, 12; Compound 3, 13; and Compound 4, 19. That the uptake process involves an estrogen-specific binder of limited capacity is demonstrated by the suppressive effect of coadministered unlabeled estradiol on target tissue uptake.

  13. Targeting matrix metalloproteinases: exploring the dynamics of the s1' pocket in the design of selective, small molecule inhibitors.

    PubMed

    Fabre, Benjamin; Ramos, Ana; de Pascual-Teresa, Beatriz

    2014-12-26

    Matrix metalloproteinases (MMPs) are important targets for pathological conditions such as arthritis, chronic obstructive pulmonary disease, and cancer. The failure of the first broad-spectrum MMP inhibitors in clinical trials has led researchers to address the selectivity as one of their main objectives. The S1' pocket has been widely used to modulate the selectivity of these enzymes because it displays the highest variability in length and shape among MMPs. In this review, we encourage medicinal chemists to also consider the dynamics of this pocket as an important parameter to achieve the desired selectivity. To support this proposal, we collect examples from the literature where the flexibility of the S1' pocket was highlighted as a relevant and significant issue affecting selectivity. We also review the experimental studies on the dynamics of this pocket. PMID:25265401

  14. Saccadic Target Selection Deficits after Lateral Intraparietal Area Inactivation in Monkeys

    E-print Network

    Dominey, Peter F.

    or memorized targets, saccades to syn- chronous and asynchronous bilateral targets, and visual search., 2002) as well as motor signals in relation to the planning and execution of saccades (Barash et al is directly involved in the representation of motor plans for saccades (Mazzoni et al., 1996). Although

  15. Identified target-selective visual interneurons descending from the dragonfly brain

    Microsoft Academic Search

    Robert M. Olberg

    1986-01-01

    1.Eight large interneurons descending in the dragonfly (Aeshna umbrosa, Anax junius) ventral nerve cord from the brain to the thoracic ganglia were identified anatomically with intracellular dye injection (Fig. 3). All eight were strictly visual and responded only to movements of small patterns, such as black squares, ‘targets’, moving on a white background.2.The target interneurons all projected from the protocerebrum

  16. Saccade target selection in macaque during feature and conjunction visual search

    Microsoft Academic Search

    NARCISSE P. BICHOT; JEFFREY D. SCHALL

    1999-01-01

    To gain insight into how vision guides eye movements, monkeys were trained to make a single saccade to a specified target stimulus during feature and conjunction search with stimuli discriminated by color and shape. Monkeys performed both tasks at levels well above chance. The latencies of saccades to the target in conjunction search exhibited shallow positive slopes as a function

  17. Going beyond: Target selection and mission analysis of human exploration missions to Near-Earth Asteroids

    NASA Astrophysics Data System (ADS)

    Zimmer, A. K.; Messerschmid, E.

    2011-12-01

    Missions to Near-Earth Asteroids (NEAs) offer a wide range of possibilities for space exploration, scientific research, and technology demonstration. In particular, manned missions to NEAs provide a unique opportunity to be the first human expedition to an interplanetary body beyond the Earth-Moon system and represent the perfect environment to gain experience in deep-space operations, which is an indispensable prerequisite for human missions to Mars. As a starting point for the analysis of such missions, the objectives of this study are to identify target asteroids and evaluate possible transfer trajectories as well as the associated launch windows. The list of accessible asteroids is narrowed down by taking dynamical and structural properties such as size and rotation rate into account. An accessibility model for NEAs is developed allowing pre-selection of asteroid targets for human missions. For this model, a novel approach is taken which assesses the accessibility of a NEA not by considering its orbital parameters separately. Instead, accessibility is determined by evaluating the combination of all orbital parameters only limited by mission duration (less than 365 days) and round-trip ?v (less than 10 km/s). In order to verify the reliability of the model, mission architectures for missions departing from low-Earth orbit are investigated and transfers to 2567 NEAs in the time frame from 2020 to 2040 are simulated. Two hundred and forty asteroids are found to be accessible for human missions under the given boundary conditions and are observed to nicely fit the model developed. Seventy three of these remaining asteroids can be reached with a ?v?7.5km/s, 15 of which allow mission durations of less than 200 days. One hundred and seventy launch windows strongly varying in duration are found for these 73 asteroids between 2020 and 2040. Launch opportunity analysis shows that several launch windows open every year in the given time frame for missions with durations of less than 365 days and ?v?7.5km/s. Although these launch windows are not evenly spaced and tend to cluster, the frequency and width of most launch windows is sufficient for a sustainable campaign. An example campaign with seven missions between 2025 and 2038 and two additional optional missions is provided. The strategy for this campaign is to gradually increase mission duration along with the time spent in the vicinity of the asteroid in order to induce a constant development of more advanced technologies at a manageable risk following a stepping stone approach and paving the way for human exploration missions to Mars. Lastly, the possibilities of mission abort are examined considering a free return scenario and an anytime abort. The free return option, characterized by a long return duration and a low ?v, is found to be feasible for all missions. The anytime abort, allowing a comparatively fast return to Earth at a ?v penalty, is observed to be an option only on short missions. Which abort scenarios are possible on a certain mission should be studied on a case-by-case basis. With these results, the mission analysis of the interplanetary part of human missions to asteroids is concluded, setting mission-specific requirements and boundary conditions required for subsequent spacecraft design.

  18. Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation

    PubMed Central

    Seydoux, Emilie; Rothen-Rutishauser, Barbara; Nita, Izabela M; Balog, Sandor; Gazdhar, Amiq; Stumbles, Philip A; Petri-Fink, Alke; Blank, Fabian; von Garnier, Christophe

    2014-01-01

    Introduction Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods Bone marrow–derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4+ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results The frequency of PS particle–positive CD11c+/CD11b+ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4+ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4+ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles. PMID:25152619

  19. Nematode control in spring-born suckler beef calves using targeted selective anthelmintic treatments.

    PubMed

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

    2014-09-15

    As anthelmintic resistance is increasingly being reported in cattle worldwide, there is a need to explore alternative approaches to gastrointestinal nematode control in cattle. A novel approach is the use of targeted selective treatments (TST) where only individual animals are treated instead of the entire group. The study objective was to determine if anthelmintic usage could be reduced using a TST-based approach in rotationally grazed first-grazing season suckler beef calves without affecting calf performance. Eighty-eight spring-born suckler beef calves, naïve to anthelmintics, with an initial mean (s.d.) age and live weight of 159 (22.4) days and 221 (42.4) kg, respectively, were used. All calves were vaccinated at pasture against dictyocaulosis at 8 and 12 weeks old. On August 9th 2013 (Week 0), when the trial began, calves were randomised by age, weight, sex, dam breed and sire breed to one of two treatments: (1) standard treatment (positive control) (n=44) and (2) TST (n=44). Samples collected one week prior to the start of the study were used as baseline covariates. Each treatment group was replicated once. All calves in the control groups were treated subcutaneously with levamisole on Week 0 and on Week 6. Individual calves in the TST groups were only eligible for treatment at pasture with the same product if predetermined thresholds were reached [plasma pepsinogen ? 2.0 international units of tyrosine/litre and faecal egg count ? 200 eggs per gram of faeces]. The trial concluded at housing on Week 13. Data were analysed using repeated measures mixed models ANOVA (PROC MIXED) (SAS 9.3). No calves in the TST groups were treated for gastrointestinal nematodes during the study period as they did not reach pre-determined treatment thresholds. Mean (sem) calf daily live weight gain for control and TST groups was 0.90 (±0.04) and 0.92 (±0.03) kg, respectively (P=0.68). Using an ELISA to detect antibodies to Dictyocaulus viviparus at Week 11, 81% of calves were seropositive. Gastrointestinal nematode challenge in spring-born suckler beef calves under these conditions can potentially be controlled with minimal anthelmintic treatments whilst not significantly impairing calf performance, provided appropriate control measures are taken to prevent dictyocaulosis from occurring. PMID:25085771

  20. Lysosomal storage disorders: Molecular basis and laboratory testing

    PubMed Central

    2011-01-01

    Lysosomal storage disorders (LSDs) are a large group of more than 50 different inherited metabolic diseases which, in the great majority of cases, result from the defective function of specific lysosomal enzymes and, in cases, of non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biogenesis. The progressive lysosomal accumulation of undegraded metabolites results in generalised cell and tissue dysfunction, and, therefore, multi-systemic pathology. Storage may begin during early embryonic development, and the clinical presentation for LSDs can vary from an early and severe phenotype to late-onset mild disease. The diagnosis of most LSDs--after accurate clinical/paraclinical evaluation, including the analysis of some urinary metabolites--is based mainly on the detection of a specific enzymatic deficiency. In these cases, molecular genetic testing (MGT) can refine the enzymatic diagnosis. Once the genotype of an individual LSD patient has been ascertained, genetic counselling should include prediction of the possible phenotype and the identification of carriers in the family at risk. MGT is essential for the identification of genetic disorders resulting from non-enzymatic lysosomal protein defects and is complementary to biochemical genetic testing (BGT) in complex situations, such as in cases of enzymatic pseudodeficiencies. Prenatal diagnosis is performed on the most appropriate samples, which include fresh or cultured chorionic villus sampling or cultured amniotic fluid. The choice of the test--enzymatic and/or molecular--is based on the characteristics of the defect to be investigated. For prenatal MGT, the genotype of the family index case must be known. The availability of both tests, enzymatic and molecular, enormously increases the reliability of the entire prenatal diagnostic procedure. To conclude, BGT and MGT are mostly complementary for post- and prenatal diagnosis of LSDs. Whenever genotype/phenotype correlations are available, they can be helpful in predicting prognosis and in making decisions about therapy. PMID:21504867

  1. Oncogene-targeting T cells reject large tumors, while oncogene inactivation selects escape variants in mouse models of cancer

    PubMed Central

    Anders, Kathleen; Buschow, Christian; Herrmann, Andreas; Milojkovic, Ana; Loddenkemper, Christoph; Kammertoens, Thomas; Daniel, Peter; Yu, Hua; Charo, Jehad; Blankenstein, Thomas

    2013-01-01

    SUMMARY The genetic instability of cancer cells frequently causes drug resistance. We established mouse cancer models, which allowed targeting of an oncogene by drug-mediated inactivation or mono-specific CD8+ effector T (TE) cells. Drug treatment of genetically-unstable large tumors was effective but selected resistant clones in the long term. In contrast, TE cells completely rejected large tumors (?500 mm3), if the target antigen was cancer-driving and expressed in sufficient amounts. While drug-mediated oncogene inactivation selectively killed the cancer cells and left the tumor vasculature intact, which likely facilitated survival and growth of resistant clones, TE cell treatment led to blood vessel destruction and probably “bystander” elimination of escape variants, which did not require antigen cross-presentation by stromal cells. PMID:22172721

  2. Oncogene-targeting T cells reject large tumors while oncogene inactivation selects escape variants in mouse models of cancer.

    PubMed

    Anders, Kathleen; Buschow, Christian; Herrmann, Andreas; Milojkovic, Ana; Loddenkemper, Christoph; Kammertoens, Thomas; Daniel, Peter; Yu, Hua; Charo, Jehad; Blankenstein, Thomas

    2011-12-13

    The genetic instability of cancer cells frequently causes drug resistance. We established mouse cancer models, which allowed targeting of an oncogene by drug-mediated inactivation or monospecific CD8(+) effector T (T(E)) cells. Drug treatment of genetically unstable large tumors was effective but selected resistant clones in the long term. In contrast, T(E) cells completely rejected large tumors (?500 mm(3)), if the target antigen was cancer-driving and expressed in sufficient amounts. Although drug-mediated oncogene inactivation selectively killed the cancer cells and left the tumor vasculature intact, which likely facilitated survival and growth of resistant clones, T(E) cell treatment led to blood vessel destruction and probably "bystander" elimination of escape variants, which did not require antigen cross-presentation by stromal cells. PMID:22172721

  3. A minimally invasive multifunctional nanoscale system for selective targeting, imaging, and NIR photothermal therapy of malignant tumors

    NASA Astrophysics Data System (ADS)

    Green, H. N.; Martyshkin, D. V.; Rosenthal, E. L.; Mirov, S. B.

    2011-03-01

    The anti-EGFR antibody, cetuximab, was labeled with IRDye 800CW fluorescent dye and conjugated to gold nanorods (GNR). GNR with aspect ratio of ~ 4 and plasmon resonance peak at ~785 nm were fabricated for use in these experiments. The IRDye:cetuximab:nanorod conjugate treatment with NIR light selectively heated the GNR and was sufficient to treat cancers. Excitation induced fluorescence of the IRDye 800CW enabling real-time imaging. We characterized and optimized the parameters for the conjugation of the GNR to cetuximab to facilitate active targeting of the nanorods to the site of the tumor. This combination of selective targeting, imaging, and photothermal treating of malignant cells is a viable approach for a variety of squamous cell carcinomas.

  4. Combination therapies for lysosomal storage disease: is the whole greater than the sum of its parts?

    PubMed Central

    Hawkins-Salsbury, Jacqueline A.; Reddy, Adarsh S.; Sands, Mark S.

    2011-01-01

    Lysosomal storage diseases (LSDs), as a group, are among the most common inherited diseases affecting children. The primary defect is typically a genetic deficiency of one of the lysosomal enzymes, often causing accumulation of undegraded substrates within the lysosome. This accumulation causes numerous secondary effects that contribute to the disease phenotype. Viral-mediated gene therapy (GT) can supply a persistent source of the deficient enzyme. However, with some notable exceptions, GT has been only modestly successful as a single approach. Recently, various therapies have been combined in order to more effectively target the diverse pathogenic mechanisms at work in LSDs. One strategy that has shown promise involves providing a persistent source of the deficient enzyme (GT, stem cell transplantation) while targeting a secondary consequence of disease with a more transient approach (substrate reduction, anti-inflammatories, pharmacological mimetic, etc.). This general strategy has resulted in both additive and synergistic effects. Interestingly, some therapeutic approaches by themselves provide essentially no clinical benefit but contribute greatly to the overall efficacy when used in combination with other treatments. Unfortunately, no therapeutic combination is universally effective. This adds to the difficulty in predicting and identifying combinations that will be most effective for individual LSDs. A better understanding of both pathogenic and therapeutic mechanisms is necessary in order to identify potentially successful combinations. While a single treatment would be ideal, the complex nature of these diseases may unavoidably limit the efficacy of single therapies. In order to more successfully treat LSDs, a shift in focus towards a combination therapy may be necessary. PMID:21421999

  5. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase

    PubMed Central

    Deacon, Sean W.; Beeser, Alexander; Fukui, Jami A.; Rennefahrt, Ulrike E. E.; Myers, Cynthia; Chernoff, Jonathan; Peterson, Jeffrey R.

    2015-01-01

    SUMMARY Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, pre-activated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate a novel strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak. PMID:18420139

  6. Cancer-Selective Targeting of the NF-?B Survival Pathway with GADD45?/MKK7 Inhibitors

    PubMed Central

    Tornatore, Laura; Sandomenico, Annamaria; Raimondo, Domenico; Low, Caroline; Rocci, Alberto; Tralau-Stewart, Cathy; Capece, Daria; D’Andrea, Daniel; Bua, Marco; Boyle, Eileen; van Duin, Mark; Zoppoli, Pietro; Jaxa-Chamiec, Albert; Thotakura, Anil K.; Dyson, Julian; Walker, Brian A.; Leonardi, Antonio; Chambery, Angela; Driessen, Christoph; Sonneveld, Pieter; Morgan, Gareth; Palumbo, Antonio; Tramontano, Anna; Rahemtulla, Amin; Ruvo, Menotti; Franzoso, Guido

    2014-01-01

    Summary Constitutive NF-?B signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-?B-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-?B-regulated antiapoptotic factor GADD45? and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45?/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-?B pathway is possible and, at least for myeloma patients, promises a profound benefit. PMID:25314077

  7. Cancer-selective targeting of the NF-?B survival pathway with GADD45?/MKK7 inhibitors.

    PubMed

    Tornatore, Laura; Sandomenico, Annamaria; Raimondo, Domenico; Low, Caroline; Rocci, Alberto; Tralau-Stewart, Cathy; Capece, Daria; D'Andrea, Daniel; Bua, Marco; Boyle, Eileen; van Duin, Mark; Zoppoli, Pietro; Jaxa-Chamiec, Albert; Thotakura, Anil K; Dyson, Julian; Walker, Brian A; Leonardi, Antonio; Chambery, Angela; Driessen, Christoph; Sonneveld, Pieter; Morgan, Gareth; Palumbo, Antonio; Tramontano, Anna; Rahemtulla, Amin; Ruvo, Menotti; Franzoso, Guido

    2014-10-13

    Constitutive NF-?B signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-?B-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-?B-regulated antiapoptotic factor GADD45? and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45?/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-?B pathway is possible and, at least for myeloma patients, promises a profound benefit. PMID:25314077

  8. K-targeted metabolomic analysis extends chemical subtraction to DESIGNER extracts: selective depletion of extracts of hops (Humulus lupulus).

    PubMed

    Ramos Alvarenga, René F; Friesen, J Brent; Nikoli?, Dejan; Simmler, Charlotte; Napolitano, José G; van Breemen, Richard; Lankin, David C; McAlpine, James B; Pauli, Guido F; Chen, Shao-Nong

    2014-12-26

    This study introduces a flexible and compound targeted approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources, generating DESIGNER extracts, by means of chemical subtraction or augmentation of metabolites. Targeting metabolites based on their liquid-liquid partition coefficients (K values), K targeting uses countercurrent separation methodology to remove single or multiple compounds from a chemically complex mixture, according to the following equation: DESIGNER extract = total extract ± target compound(s). Expanding the scope of the recently reported depletion of extracts by immunoaffinity or solid phase liquid chromatography, the present approach allows a more flexible, single- or multi-targeted removal of constituents from complex extracts such as botanicals. Chemical subtraction enables both chemical and biological characterization, including detection of synergism/antagonism by both the subtracted targets and the remaining metabolite mixture, as well as definition of the residual complexity of all fractions. The feasibility of the DESIGNER concept is shown by K-targeted subtraction of four bioactive prenylated phenols, isoxanthohumol (1), 8-prenylnaringenin (2), 6-prenylnaringenin (3), and xanthohumol (4), from a standardized hops (Humulus lupulus L.) extract using specific solvent systems. Conversely, adding K-targeted isolates allows enrichment of the original extract and hence provides an augmented DESIGNER material. Multiple countercurrent separation steps were used to purify each of the four compounds, and four DESIGNER extracts with varying depletions were prepared. The DESIGNER approach innovates the characterization of chemically complex extracts through integration of enabling technologies such as countercurrent separation, K-by-bioactivity, the residual complexity concepts, as well as quantitative analysis by (1)H NMR, LC-MS, and HiFSA-based NMR fingerprinting. PMID:25437744

  9. The Retinoblastoma Protein Selectively Represses E2F1 Targets via a TAAC DNA Element during Cellular Senescence*

    PubMed Central

    Chen, Tianda; Xue, Lixiang; Niu, Jing; Ma, Liwei; Li, Na; Cao, Xiaoxiao; Li, Qian; Wang, Meng; Zhao, Wenting; Li, Guodong; Wang, Jiamu; Tong, Tanjun

    2012-01-01

    The retinoblastoma (Rb) protein mediates heterochromatin formation at the promoters of E2 transcription factor 1 (E2F1) target genes, such as proliferating cell nuclear antigen and cyclin A2 (CCNA2), and represses these genes during cellular senescence. However, the selectivity of Rb recruitment is still not well understood. Here, we demonstrate that a senescence-associated gene is a direct target of E2F1 and is also repressed by heterochromatin in senescent cells. In contrast, ARF and p27KIP1, which are also E2F1 targets, are not repressed by Rb and heterochromatin formation. By comparing the promoter sequences of these genes, we found a novel TAAC element that is present in the cellular senescence-inhibited gene, proliferating cell nuclear antigen, and CCNA2 promoters but absent from the ARF and p27KIP1 promoters. This TAAC element associates with Rb and is required for Rb recruitment. We further determined that TAAC element-mediated Rb association requires the E2F1 binding site, but not E2F1 protein. These results provide a novel molecular mechanism for the different expression patterns of E2F1 targets and afford new mechanistic insight regarding the selectivity of Rb-mediated heterochromatin formation and gene repression during cellular senescence. PMID:22955272

  10. The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death

    PubMed Central

    Qiao, Shuxi; Tao, Shasha; Rojo de la Vega, Montserrat; Park, Sophia L; Vonderfecht, Amanda A; Jacobs, Suesan L; Zhang, Donna D; Wondrak, Georg T

    2013-01-01

    Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A, E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells. PMID:24113242

  11. Selection and optimization of gene targets for the metabolic engineering of E. coli

    E-print Network

    Fischer, Curt R., Ph. D. Massachusetts Institute of Technology

    2009-01-01

    This thesis is about identifying genetic interventions that improve the performance of targeted pathways in the metabolism of the bacterium Escherichia coli. Three case studies illustrate three disparate approaches to ...

  12. Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells

    E-print Network

    Fink, Brian D.; O'Malley, Yunxia; Dake, Brian L.; Ross, Nicholette C.; Prisinzano, Thomas E.; Sivitz, William I.

    2009-01-22

    but not complex II substrates. Methods and Results To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone...

  13. Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis

    E-print Network

    Farhat, Maha R

    M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely ...

  14. The Dendritic Cell Receptor for Endocytosis, DEC205, Can Recycle and Enhance Antigen Presentation via Major Histocompatibility Complex Class II-positive Lysosomal Compartments

    Microsoft Academic Search

    Karsten Mahnke; Ming Guo; Sena Lee; Homero Sepulveda; Suzy L. Swain; Michel Nussenzweig; Ralph M. Steinman

    2000-01-01

    Many receptors for endocytosis recycle into and out of cells through early endosomes. We now find in dendritic cells that the DEC-205 multilectin receptor targets late endosomes or lysosomes rich in major his- tocompatibility complex class II (MHC II) products, whereas the homologous macrophage mannose recep- tor (MMR), as expected, is found in more peripheral endosomes. To analyze this finding,

  15. Receptor binding peptides for target-selective delivery of nanoparticles encapsulated drugs

    PubMed Central

    Accardo, Antonella; Aloj, Luigi; Aurilio, Michela; Morelli, Giancarlo; Tesauro, Diego

    2014-01-01

    Active targeting by means of drug encapsulated nanoparticles decorated with targeting bioactive moieties represents the next frontier in drug delivery; it reduces drug side effects and increases the therapeutic index. Peptides, based on their chemical and biological properties, could have a prevalent role to direct drug encapsulated nanoparticles, such as liposomes, micelles, or hard nanoparticles, toward the tumor tissues. A considerable number of molecular targets for peptides are either exclusively expressed or overexpressed on both cancer vasculature and cancer cells. They can be classified into three wide categories: integrins; growth factor receptors (GFRs); and G-protein coupled receptors (GPCRs). Therapeutic agents based on nanovectors decorated with peptides targeting membrane receptors belonging to the GPCR family overexpressed by cancer cells are reviewed in this article. The most studied targeting membrane receptors are considered: somatostatin receptors; cholecystokinin receptors; receptors associated with the Bombesin like peptides family; luteinizing hormone-releasing hormone receptors; and neurotensin receptors. Nanovectors of different sizes and shapes (micelles, liposomes, or hard nanoparticles) loaded with doxorubicin or other cytotoxic drugs and externally functionalized with natural or synthetic peptides are able to target the overexpressed receptors and are described based on their formulation and in vitro and in vivo behaviors. PMID:24741304

  16. Peptidic Tumor Targeting Agents: The Road from Phage Display Peptide Selections to Clinical Applications

    PubMed Central

    Brown, Kathlynn C.

    2014-01-01

    Cancer has become the number one cause of death amongst Americans, killing approximately 1,600 people per day. Novel methods for early detection and the development of effective treatments are an eminent priority in medicine. For this reason, isolation of tumor-specific ligands is a growing area of research. Tumor-specific binding agents can be used to probe the tumor cell surface phenotype and customize treatment accordingly by conjugating the appropriate cell-targeting ligand to an anticancer drug. This refines the molecular diagnosis of the tumor and creates guided drugs that can target the tumor while sparing healthy tissues. Additionally, these targeting agents can be used as in vivo imaging agents that allow for earlier detection of tumors and micrometastasis. Phage display is a powerful technique for the isolation of peptides that bind to a particular target with high affinity and specificity. The biopanning of intact cancer cells or tumors in animals can be used to isolate peptides that bind to cancer-specific cell surface biomarkers. Over the past 10 years, unbiased biopanning of phage-displayed peptide libraries has generated a suite of cancer targeting peptidic ligands. This review discusses the recent advances in the isolation of cancer-targeting peptides by unbiased biopanning methods and highlights the use of the isolated peptides in clinical applications. PMID:20030617

  17. Transient analysis mode participation for modal survey target mode selection using MSC/NASTRAN DMAP

    NASA Technical Reports Server (NTRS)

    Barnett, Alan R.; Ibrahim, Omar M.; Sullivan, Timothy L.; Goodnight, Thomas W.

    1994-01-01

    Many methods have been developed to aid analysts in identifying component modes which contribute significantly to component responses. These modes, typically targeted for dynamic model correlation via a modal survey, are known as target modes. Most methods used to identify target modes are based on component global dynamic behavior. It is sometimes unclear if these methods identify all modes contributing to responses important to the analyst. These responses are usually those in areas of hardware design concerns. One method used to check the completeness of target mode sets and identify modes contributing significantly to important component responses is mode participation. With this method, the participation of component modes in dynamic responses is quantified. Those modes which have high participation are likely modal survey target modes. Mode participation is most beneficial when it is used with responses from analyses simulating actual flight events. For spacecraft, these responses are generated via a structural dynamic coupled loads analysis. Using MSC/NASTRAN DMAP, a method has been developed for calculating mode participation based on transient coupled loads analysis results. The algorithm has been implemented to be compatible with an existing coupled loads methodology and has been used successfully to develop a set of modal survey target modes.

  18. Inhibition of lysosome degradation on autophagosome formation and responses to GMI, an immunomodulatory protein from Ganoderma microsporum

    PubMed Central

    Hsin, I-Lun; Sheu, Gwo-Tarng; Jan, Ming-Shiou; Sun, Hai-Lun; Wu, Tzu-Chin; Chiu, Ling-Yen; Lue, Ko-Huang; Ko, Jiunn-Liang

    2012-01-01

    BACKGROUND AND PURPOSE Autophagic cell death is considered a self-destructive process that results from large amounts of autophagic flux. In our previous study, GMI, a recombinant fungal immunomodulatory protein cloned from Ganoderma microsporum, induced autophagic cell death in lung cancer cells. The aim of this study was to examine the role of autophagosome accumulation in GMI-mediated cell death. EXPERIMENTAL APPROACH Western blot analysis, flow cytometry and confocal microscopy were used to evaluate the effects of different treatments, including silencing of ATP6V0A1 by use of short hairpin RNAi, on GMI-mediated cell death, lung cancer cell viability and autophagosome accumulation in vitro. KEY RESULTS Lysosome inhibitors bafilomycin-A1 and chloroquine increased GMI-mediated autophagic cell death. GMI and bafilomycin-A1 co-treatment induced the accumulation of large amounts of autophagosomes, but did not significantly induce apoptosis. GMI elicited autophagy through the PKB (Akt)/mammalian target of rapamycin signalling pathway. Silencing of ATP6V0A1, one subunit of vesicular H+-ATPases (V-ATPases) that mediates lysosome acidification, spontaneously induced autophagosome accumulation, but did not affect lysosome acidity. GMI-mediated autophagosome accumulation and cytotoxicity was increased in shATP6V0A1 lung cancer cells. Furthermore, ATP6V0A1 silencing decreased autophagosome and lysosome fusion in GMI-treated CaLu-1/GFP-LC3 lung cancer cells. CONCLUSION AND IMPLICATIONS We demonstrated that autophagosome accumulation induces autophagic cell death in a GMI treatment model, and ATP6V0A1 plays an important role in mediating autophagosome-lysosome fusion. Our findings provide new insights into the mechanisms involved in the induction of autophagic cell death. PMID:22708544

  19. Fig4 Deficiency: A Newly Emerged Lysosomal Storage Disorder?

    PubMed Central

    Martyn, Colin; Li, Jun

    2012-01-01

    FIG4 (Sac3 in mammals) is a 5’-phosphoinositide phosphatase that coordinates the turnover of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), a very low abundance phosphoinositide. Deficiency of FIG4 severely affects the human and mouse nervous systems by causing two distinct forms of abnormal lysosomal storage. The first form occurs in spinal sensory neurons, where vacuolated endolysosomes accumulate in perinuclear regions. A second form occurs in cortical/spinal motor neurons and glia, in which enlarged endolysosomes become filled with electron dense materials in a manner indistinguishable from other lysosomal storage disorders. Humans with a deficiency of FIG4 (known as Charcot-Marie-Tooth disease type 4J or CMT4J) present with clinical and pathophysiological phenotypes indicative of spinal motor neuron degeneration and segmental demyelination. These findings reveal a signaling pathway involving FIG4 that appears to be important for lysosomal function. In this review, we discuss the biology of FIG4 and describe how the deficiency of FIG4 results in lysosomal phenotypes. We also discuss the implications of FIG4/PI(3,5)P2 signaling in understanding other lysosomal storage diseases, neuropathies, and acquired demyelinating diseases. PMID:23165282

  20. Autophagy, mitochondria and cell death in lysosomal storage diseases

    PubMed Central

    Kiselyov, Kirill; Jennigs, John J.; Rbaibi, Youssef; Chu, Charleen T.

    2009-01-01

    Lysosomal storage diseases (LSDs) are debilitating genetic conditions that frequently manifest as neurodegenerative disorders. They severely affect eye, motor and cognitive functions and, in most cases, abbreviate the lifespan. Postmitotic cells such as neurons and mononuclear phagocytes rich in lysosomes are most often affected by the accumulation of undegraded material. Cell death is well documented in parts of the brain and in other cells of LSD patients and animal models, although little is known about mechanisms by which death pathways are activated in these diseases, and not all cells exhibiting increased storage material are affected by cell death. Lysosomes are essential for maturation and completion of autophagy-initiated protein and organelle degradation. Moreover, accumulation of effete mitochondria has been documented in postmitotic cells whose lysosomal function is suppressed or in aging cells with lipofuscin accumulation. Based upon observations in the literature and our own data showing similar mitochondrial abnormalities in several LSDs, we propose a new model of cell death in LSDs. We suggest that the lysosomal deficiencies in LSDs inhibit autophagic maturation, leading to a condition of autophagic stress. The resulting accumulation of dysfunctional mitochondria showing impaired Ca2+ buffering increases the vulnerability of the cells to pro-apoptotic signals. PMID:17329960

  1. ATP Release through Lysosomal Exocytosis from Peripheral Nerves: The Effect of Lysosomal Exocytosis on Peripheral Nerve Degeneration and Regeneration after Nerve Injury

    PubMed Central

    Jung, Junyang; Kwon, Hyunseob

    2014-01-01

    Studies have shown that lysosomal activation increases in Schwann cells after nerve injury. Lysosomal activation is thought to promote the engulfment of myelin debris or fragments of injured axons in Schwann cells during Wallerian degeneration. However, a recent interpretation of lysosomal activation proposes a different view of the phenomenon. During Wallerian degeneration, lysosomes become secretory vesicles and are activated for lysosomal exocytosis. The lysosomal exocytosis triggers adenosine 5?-triphosphate (ATP) release from peripheral neurons and Schwann cells during Wallerian degeneration. Exocytosis is involved in demyelination and axonal degradation, which facilitate nerve regeneration following nerve degeneration. At this time, released ATP may affect the communication between cells in peripheral nerves. In this review, our description of the relationship between lysosomal exocytosis and Wallerian degeneration has implications for the understanding of peripheral nerve degenerative diseases and peripheral neuropathies, such as Charcot-Marie-Tooth disease or Guillain-Barré syndrome. PMID:25101301

  2. Classifier-based offline feature selection and evaluation for visual tracking of sea-surface and aerial targets

    NASA Astrophysics Data System (ADS)

    Çak?r, Serdar; Aytaç, Tayfun; Y?ld?r?m, Alper; Gerek, Ö. Nezih

    2011-10-01

    An offline feature selection and evaluation mechanism is used in order to develop a robust visual tracking scheme for sea-surface and aerial targets. The covariance descriptors, known to constitute an efficient signature set in object detection and classification problems, are used in the feature extraction phase of the proposed scheme. The performance of feature sets are compared using support vector machines, and those resulting in the highest detection performance are used in the covariance based tracker. The tracking performance is evaluated in different scenarios using different performance measures with respect to ground truth target positions. The proposed tracking scheme is observed to track sea-surface and aerial targets with plausible accuracies, and the results show that gradient-based features, together with the pixel locations and intensity values, provide robust target tracking in both surveillance scenarios. The performance of the proposed tracking strategy is also compared with some well-known trackers including correlation, Kanade-Lucas-Tomasi feature, and scale invariant feature transform-based trackers. Experimental results and observations show that the proposed target tracking scheme outperforms other trackers in both air and sea surveillance scenarios.

  3. Diagnosis of lysosomal storage disorders: Gaucher disease.

    PubMed

    Johnson, Britt A; Dajnoki, Angela; Bodamer, Olaf

    2014-01-01

    Gaucher Disease (GD) is a progressive lysosomal storage disorder caused by deficiency of glucocerebrosidase (GBA). The clinical phenotype follows a spectrum ranging from severe early-onset to milder late-onset disease. The absence of neurological involvement defines GD type I, whereas neuronopathic features define GD type II and III. Early diagnosis may be important for timely initiation of enzyme replacement therapy to prevent disease complications, although the enzyme does not cross the blood brain barrier. Diagnosis of GD can be readily achieved by analysis of GBA in leukocytes, fibroblasts, and/or dried blood spots using fluorometric, microfluidic or mass spectrometry-based assays. Low GBA activities are typically confirmed through molecular analysis of the GBA gene. GBA analysis in dried blood spots may be attractive for high-throughput screening of at-risk individuals and/or newborn infants. The method detailed in this unit is based on GBA analysis by tandem mass spectrometry following incubation of dried blood spots with the GBA-specific substrate D-glucosyl-?1-1'-N-dodecanoyl-D-erythro-sphingosine [C12-glucocerebroside (C36 H69 NO8 )] and internal standard N-myristoyl-D-erythro-sphingosine [C14-ceramide (C32 H63 NO3 )]. GBA activities in more than 2,000 newborn infants showed a mean of 22.0 ± 13.8 ?mol/hr/liter (median: 19.9 ?mol/hr/liter; 95% CI: 21.41-22.59 ?mol/hr/liter). GBA activities in an adult population (n >1,200) showed generally lower enzyme activities than newborns, with a mean of 9.87 ± 9.35 ?mol/hr/liter (median: 8.06 ?mol/hr/liter). GBA activities in ten adult patients with confirmed GD were less than 4.2 ?mol/hr/liter and in seven infants and children with GD less than 1.24 ?mol/hr/liter. This method is robust, sensitive, and suitable for high-throughput analysis of hundreds of samples. Curr. Protoc. Hum. Genet. 82:17.15.1-17.15.6. © 2014 by John Wiley & Sons, Inc. PMID:25042717

  4. Identification and characterization of alphavirus M1 as a selective oncolytic virus targeting ZAP-defective human cancers.

    PubMed

    Lin, Yuan; Zhang, Haipeng; Liang, Jiankai; Li, Kai; Zhu, Wenbo; Fu, Liwu; Wang, Fang; Zheng, Xiaoke; Shi, Huijuan; Wu, Sihan; Xiao, Xiao; Chen, Lijun; Tang, Lipeng; Yan, Min; Yang, Xiaoxiao; Tan, Yaqian; Qiu, Pengxin; Huang, Yijun; Yin, Wei; Su, Xinwen; Hu, Haiyan; Hu, Jun; Yan, Guangmei

    2014-10-21

    Oncolytic virotherapy is a growing treatment modality that uses replicating viruses as selective antineoplastic agents. Safety and efficacy considerations dictate that an ideal oncolytic agent would discriminate between normal and cancer cells on the basis of common genetic abnormalities in human cancers. Here, we identify a naturally occurring alphavirus (M1) as a novel selective killer targeting zinc-finger antiviral protein (ZAP)-deficient cancer cells. In vitro, in vivo, and ex vivo studies showed potent oncolytic efficacy and high tumor tropism of M1. We showed that the selectivity depends on ZAP deficiency by systematic identification. A large-scale multicenter pathology study using tissue microarrays reveals that ZAP is commonly deficient in human cancers, suggesting extensive application prospects for M1. Additionally, M1 killed cancer cells by inducing endoplasmic reticulum stress-mediated apoptosis. Our report provides novel insights into potentially personalized cancer therapy using oncolytic viruses. PMID:25288727

  5. Lysosomal Two-pore Channel Subtype 2 (TPC2) Regulates Skeletal Muscle Autophagic Signaling*

    PubMed Central

    Lin, Pei-Hui; Duann, Pu; Komazaki, Shinji; Park, Ki Ho; Li, Haichang; Sun, Mingzhai; Sermersheim, Mathew; Gumpper, Kristyn; Parrington, John; Galione, Antony; Evans, A. Mark; Zhu, Michael X.; Ma, Jianjie

    2015-01-01

    Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2?/? mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis. PMID:25480788

  6. Hippocampal endosomal, lysosomal, and autophagic dysregulation in mild cognitive impairment: correlation with a? and tau pathology.

    PubMed

    Perez, Sylvia E; He, Bin; Nadeem, Muhammad; Wuu, Joanne; Ginsberg, Stephen D; Ikonomovic, Milos D; Mufson, Elliott J

    2015-04-01

    Endosomal-lysosomal and autophagic dysregulation occurs in the hippocampus in prodromal Alzheimer disease (AD), but its relationship with ?-amyloid (A?) and tau pathology remains unclear. To investigate this issue, we performed immunoblot analysis of hippocampal homogenates from cases with an antemortem clinical diagnosis of no cognitive impairment, mild cognitive impairment (MCI), and AD. Western blot analysis revealed significant increases in the acid hydrolase cathepsin D and early endosome marker rabaptin5 in the MCI group compared with AD, whereas levels of phosphorylated mammalian target of rapamycin proteins (pmTOR), total mammalian target of rapamycin (mTOR), p62, traf6, and LilrB2 were comparable across clinical groups. Hippocampal A?1-40 and A?1-42 concentrations and AT8-immunopositive neurofibrillary tangle density were not significantly different across the clinical groups. Greater cathepsin D expression was associated with global cognitive score and episodic memory score but not with mini mental state examination or advanced neuropathology criteria. These results indicate that alterations in hippocampal endosomal-lysosomal proteins in MCI are independent of tau or A? pathology. PMID:25756588

  7. Effects of Type and Strength of Force Feedback on Movement Time in a Target Selection Task

    NASA Technical Reports Server (NTRS)

    Rorie, Robert Conrad; Vu, Kim-Phuong L.; Marayong, Panadda; Robles, Jose; Strybel, Thomas Z.; Battiste, Vernol

    2013-01-01

    Future cockpits will likely include new onboard technologies, such as cockpit displays of traffic information, to help support future flight deck roles and responsibilities. These new technologies may benefit from multimodal feedback to aid pilot information processing. The current study investigated the effects of multiple levels of force feedback on operator performance in an aviation task. Participants were presented with two different types of force feedback (gravitational and spring force feedback) for a discrete targeting task, with multiple levels of gain examined for each force feedback type. Approach time and time in target were recorded. Results suggested that the two highest levels of gravitational force significantly reduced approach times relative to the lowest level of gravitational force. Spring force level only affected time in target. Implications of these findings for the design of future cockpit displays will be discussed.

  8. An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters*

    PubMed Central

    Chapel, Agnès; Kieffer-Jaquinod, Sylvie; Sagné, Corinne; Verdon, Quentin; Ivaldi, Corinne; Mellal, Mourad; Thirion, Jaqueline; Jadot, Michel; Bruley, Christophe; Garin, Jérôme; Gasnier, Bruno; Journet, Agnès

    2013-01-01

    Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions. PMID:23436907

  9. Common Risk Alleles for Inflammatory Diseases Are Targets of Recent Positive Selection

    PubMed Central

    Raj, Towfique; Kuchroo, Manik; Replogle, Joseph M.; Raychaudhuri, Soumya; Stranger, Barbara E.; De Jager, Philip L.

    2013-01-01

    Genome-wide association studies (GWASs) have identified hundreds of loci harboring genetic variation influencing inflammatory-disease susceptibility in humans. It has been hypothesized that present day inflammatory diseases may have arisen, in part, due to pleiotropic effects of host resistance to pathogens over the course of human history, with significant selective pressures acting to increase host resistance to pathogens. The extent to which genetic factors underlying inflammatory-disease susceptibility has been influenced by selective processes can now be quantified more comprehensively than previously possible. To understand the evolutionary forces that have shaped inflammatory-disease susceptibility and to elucidate functional pathways affected by selection, we performed a systems-based analysis to integrate (1) published GWASs for inflammatory diseases, (2) a genome-wide scan for signatures of positive selection in a population of European ancestry, (3) functional genomics data comprised of protein-protein interaction networks, and (4) a genome-wide expression quantitative trait locus (eQTL) mapping study in peripheral blood mononuclear cells (PBMCs). We demonstrate that loci for inflammatory-disease susceptibility are enriched for genomic signatures of recent positive natural selection, with selected loci forming a highly interconnected protein-protein interaction network. Further, we identify 21 loci for inflammatory-disease susceptibility that display signatures of recent positive selection, of which 13 also show evidence of cis-regulatory effects on genes within the associated locus. Thus, our integrated analyses highlight a set of susceptibility loci that might subserve a shared molecular function and has experienced selective pressure over the course of human history; today, these loci play a key role in influencing susceptibility to multiple different inflammatory diseases, in part through alterations of gene expression in immune cells. PMID:23522783

  10. Common risk alleles for inflammatory diseases are targets of recent positive selection.

    PubMed

    Raj, Towfique; Kuchroo, Manik; Replogle, Joseph M; Raychaudhuri, Soumya; Stranger, Barbara E; De Jager, Philip L

    2013-04-01

    Genome-wide association studies (GWASs) have identified hundreds of loci harboring genetic variation influencing inflammatory-disease susceptibility in humans. It has been hypothesized that present day inflammatory diseases may have arisen, in part, due to pleiotropic effects of host resistance to pathogens over the course of human history, with significant selective pressures acting to increase host resistance to pathogens. The extent to which genetic factors underlying inflammatory-disease susceptibility has been influenced by selective processes can now be quantified more comprehensively than previously possible. To understand the evolutionary forces that have shaped inflammatory-disease susceptibility and to elucidate functional pathways affected by selection, we performed a systems-based analysis to integrate (1) published GWASs for inflammatory diseases, (2) a genome-wide scan for signatures of positive selection in a population of European ancestry, (3) functional genomics data comprised of protein-protein interaction networks, and (4) a genome-wide expression quantitative trait locus (eQTL) mapping study in peripheral blood mononuclear cells (PBMCs). We demonstrate that loci for inflammatory-disease susceptibility are enriched for genomic signatures of recent positive natural selection, with selected loci forming a highly interconnected protein-protein interaction network. Further, we identify 21 loci for inflammatory-disease susceptibility that display signatures of recent positive selection, of which 13 also show evidence of cis-regulatory effects on genes within the associated locus. Thus, our integrated analyses highlight a set of susceptibility loci that might subserve a shared molecular function and has experienced selective pressure over the course of human history; today, these loci play a key role in influencing susceptibility to multiple different inflammatory diseases, in part through alterations of gene expression in immune cells. PMID:23522783

  11. A Framework for Spatial Feature Selection and Scoping and its Application to Geo-Targeting

    E-print Network

    Eick, Christoph F.

    77004 USA 2 lyons19@cs.uh.edu 3 ceick@cs.uh.edu + ThinkersRUS Sunnyvale, CA USA 4 abagher@thinkersr.us advertising. Associations between Internet ad performance data, such as number of clicks or Click Through Rate create valuable knowledge for geo-targeted advertising. In this paper, we present a novel framework

  12. Using market segmentation theory to select target markets for sun protection campaigns

    Microsoft Academic Search

    Sandra C. Jones; Lyn Rees; D. Hall; A. Tang

    2005-01-01

    This paper describes the initial steps in target market segmentation and evaluation as part of an industry-linked research project to develop a social marketing program for sun protection. The Project Reference Group developed a set of segmentation evaluation criteria based on recommendations from marketing and health promotion literature, as well as adding criteria specifically relevant to the industry partner. The

  13. Target Selection by the Frontal Cortex during Coordinated Saccadic and Smooth Pursuit Eye Movements

    ERIC Educational Resources Information Center

    Srihasam, Krishna; Bullock, Daniel; Grossberg, Stephen

    2009-01-01

    Oculomotor tracking of moving objects is an important component of visually based cognition and planning. Such tracking is achieved by a combination of saccades and smooth-pursuit eye movements. In particular, the saccadic and smooth-pursuit systems interact to often choose the same target, and to maximize its visibility through time. How do…

  14. A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum

    Microsoft Academic Search

    Chang Hyun Khang; Sook-Young Park; Yong-Hwan Lee; Seogchan Kang

    2005-01-01

    Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the

  15. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte

    E-print Network

    Lieberman, Judy

    of concept, a fusion protein, designed by using an Fab fragment of an anti-HIV envelope (env) antibody and validating drug targets and poten- tially for therapy. Lymphocytes and other primary blood cells-directed siRNA delivery. affinity up-regulation antiinflammation cell adhesion molecule drug delivery AL-57

  16. Selection of Intracellular Single-Domain Antibodies Targeting the HIV-1 Vpr Protein by Cytoplasmic Yeast Two-Hybrid System

    PubMed Central

    Matz, Julie; Hérate, Cécile; Bouchet, Jérôme; Dusetti, Nelson; Gayet, Odile; Baty, Daniel; Benichou, Serge; Chames, Patrick

    2014-01-01

    The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system. PMID:25436999

  17. MLL fusion protein–driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction

    PubMed Central

    Chen, Wei; Jones, Morgan; Granowicz, Eric M.; Maillard, Ivan; Hess, Jay L.

    2013-01-01

    MLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target. PMID:23900238

  18. MLL fusion protein-driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction.

    PubMed

    Muntean, Andrew G; Chen, Wei; Jones, Morgan; Granowicz, Eric M; Maillard, Ivan; Hess, Jay L

    2013-09-12

    MLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target. PMID:23900238

  19. Regulation of locomotion and motoneuron trajectory selection and targeting by the Drosophila homolog of Olig family transcription factors

    PubMed Central

    Oyallon, Justine; Apitz, Holger; Miguel-Aliaga, Irene; Timofeev, Katarina; Ferreira, Lauren; Salecker, Iris

    2012-01-01

    During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. We investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, we demonstrate that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, our findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail. PMID:22796650

  20. Regulation of locomotion and motoneuron trajectory selection and targeting by the Drosophila homolog of Olig family transcription factors.

    PubMed

    Oyallon, Justine; Apitz, Holger; Miguel-Aliaga, Irene; Timofeev, Katarina; Ferreira, Lauren; Salecker, Iris

    2012-09-15

    During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. We investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, we demonstrate that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, our findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail. PMID:22796650

  1. Drugability of extracellular targets: discovery of small molecule drugs targeting allosteric, functional, and subunit-selective sites on GPCRs and ion channels.

    PubMed

    Grigoriadis, Dimitri E; Hoare, Samuel R J; Lechner, Sandra M; Slee, Deborah H; Williams, John A

    2009-01-01

    Beginning with the discovery of the structure of deoxyribose nucleic acid in 1953, by James Watson and Francis Crick, the sequencing of the entire human genome some 50 years later, has begun to quantify the classes and types of proteins that may have relevance to human disease with the promise of rapidly identifying compounds that can modulate these proteins so as to have a beneficial and therapeutic outcome. This so called 'drugable space' involves a variety of membrane-bound proteins including the superfamily of G-protein-coupled receptors (GPCRs), ion channels, and transporters among others. The recent number of novel therapeutics targeting membrane-bound extracellular proteins that have reached the market in the past 20 years however pales in magnitude when compared, during the same timeframe, to the advancements made in the technologies available to aid in the discovery of these novel therapeutics. This review will consider select examples of extracellular drugable targets and focus on the GPCRs and ion channels highlighting the corticotropin releasing factor (CRF) type 1 and gamma-aminobutyric acid receptors, and the Ca(V)2.2 voltage-gated ion channel. These examples will elaborate current technological advancements in drug discovery and provide a prospective framework for future drug development. PMID:18800070

  2. The cytoplasmic domain mediates localization of furin to the trans- Golgi network en route to the endosomal/lysosomal system

    PubMed Central

    1994-01-01

    To investigate the mechanisms of membrane protein localization to the Golgi complex, we have examined the intracellular trafficking of epitope-tagged forms of the mammalian endopeptidase, furin, in stably transformed rat basophilic leukemia cells. Our studies show that furin is predominantly localized to the trans-Golgi network (TGN) at steady state, with smaller amounts present in intracellular vesicles. Biochemical and morphological analyses reveal that furin is progressively delivered to a lysosomal compartment, where it is degraded. Analyses of furin deletion mutants and chimeric proteins show that the cytoplasmic domain is both necessary and sufficient for localization to the TGN in various cell types. Interestingly, deletion of most of the cytoplasmic domain of furin results in a molecule that is predominantly localized to intracellular vesicles, some of which display characteristics of lysosomes. To a lesser extent, the cytoplasmically deleted molecule is also localized to the plasma membrane. These observations suggest the existence of an additional determinant for targeting to the endosomal/lysosomal system within the lumenal and/or transmembrane domains of furin. Thus, the overall pattern of trafficking and steady state localization of furin are determined by targeting information contained within more than one region of the molecule. PMID:7914893

  3. A Computational Strategy to Select Optimized Protein Targets for Drug Development toward the Control of Cancer Diseases

    PubMed Central

    Carels, Nicolas; Tilli, Tatiana; Tuszynski, Jack A.

    2015-01-01

    In this report, we describe a strategy for the optimized selection of protein targets suitable for drug development against neoplastic diseases taking the particular case of breast cancer as an example. We combined human interactome and transcriptome data from malignant and control cell lines because highly connected proteins that are up-regulated in malignant cell lines are expected to be suitable protein targets for chemotherapy with a lower rate of undesirable side effects. We normalized transcriptome data and applied a statistic treatment to objectively extract the sub-networks of down- and up-regulated genes whose proteins effectively interact. We chose the most connected ones that act as protein hubs, most being in the signaling network. We show that the protein targets effectively identified by the combination of protein connectivity and differential expression are known as suitable targets for the successful chemotherapy of breast cancer. Interestingly, we found additional proteins, not generally targeted by drug treatments, which might justify the extension of existing formulation by addition of inhibitors designed against these proteins with the consequence of improving therapeutic outcomes. The molecular alterations observed in breast cancer cell lines represent either driver events and/or driver pathways that are necessary for breast cancer development or progression. However, it is clear that signaling mechanisms of the luminal A, B and triple negative subtypes are different. Furthermore, the up- and down-regulated networks predicted subtype-specific drug targets and possible compensation circuits between up- and down-regulated genes. We believe these results may have significant clinical implications in the personalized treatment of cancer patients allowing an objective approach to the recycling of the arsenal of available drugs to the specific case of each breast cancer given their distinct qualitative and quantitative molecular traits. PMID:25625699

  4. Bi-directionally selective bone targeting delivery for anabolic and antiresorptive drugs: a novel combined therapy for osteoporosis?

    PubMed

    Liu, Jinsong; Zhang, Hualin; Dong, Yiwen; Jin, Yifan; Hu, Xiaohui; Cai, Kaiyong; Ma, Jianfeng; Wu, Gang

    2014-12-01

    Osteoporosis is a progressive systemic skeletal disease, in which the equilibrium of bone resorption and bone formation is disturbed. The drugs for osteoporosis can be divided into two categories according to their predominant effects: antiresorptive drugs and anabolic drugs. Antiresorptive drugs are designed to inhibit bone resorption and anabolic drugs are aiming to stimulate bone formation. On the other hand, most antiresorptive drugs usually decrease anabolic activities and reduce bone formation, while anabolic drugs can unintendedly increase bone resorption. Furthermore, both types of drugs show no preferential distribution in bone and can locate generally in the areas of both bone formation and bone resorption. Consequently, the non-specific interaction of these drugs with non-targeting area and cells can lead to a compromised efficacy. Combined therapies of antiresorptive and anabolic drugs do not necessarily yield superiority when compared to monotherapy. Here, basing on the targeting cells of these two kinds of drugs and the spatial distribution of osteoblasts and osteoclasts, we propose a novel drug delivery system of bi-directionally selective targeting in order to facilitate the efficacy of antiresorptive and anabolic drugs in combined therapy. In the system, an antiresorptive drug will be linked with a peptide of the eight repeating sequences of aspartate--(Asp)8 that can preferentially guide the drugs to bone resorption zone; while an anabolic drug linked with a peptide of six repeats of the sequence aspartate, serine, serine--(Asp-Ser-Ser)6 that can favorably guide the drugs to bone formation zone. The novel delivery system will improve the specific interaction between the drugs and their targeting cells. Furthermore, the system will reduce the non-specific interaction of the anabolic and antiresorptive drugs with their respective non-targeting cells, which will maximally reduce their side-effects. Therefore, we postulate that the new bone targeting drug delivery system will be a blessing for osteoporotic patients. PMID:25459136

  5. On and off-target effects of telomere uncapping G-quadruplex selective ligands based on pentacyclic acridinium salts

    PubMed Central

    2013-01-01

    Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment. The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant ?2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay. Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with ?2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities. Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment. PMID:24330541

  6. Small-Colony Mutants of Staphylococcus aureus Allow Selection of Gyrase-Mediated Resistance to Dual-Target Fluoroquinolones

    PubMed Central

    Pan, Xiao-Su; Hamlyn, Penelope J.; Talens-Visconti, Raquel; Alovero, Fabiana L.; Manzo, Ruben H.; Fisher, L. Mark

    2002-01-01

    Fluoroquinolones acting equally through DNA gyrase and topoisomerase IV in vivo are considered desirable in requiring two target mutations for emergence of resistant bacteria. To investigate this idea, we have studied the response of Staphylococcus aureus RN4220 to stepwise challenge with sparfloxacin, a known dual-target agent, and with NSFQ-105, a more potent sulfanilyl fluoroquinolone that behaves similarly. First-step mutants were obtained with both drugs but only at the MIC. These mutants exhibited distinctive small-colony phenotypes and two- to fourfold increases in MICs of NSFQ-105, sparfloxacin, and ciprofloxacin. No changes were detected in the quinolone resistance-determining regions of the gyrA, gyrB, grlA, or grlB gene. Quinolone-induced small-colony mutants shared the delayed coagulase response but not the requirement for menadione, hemin, or thymidine characteristic of small-colony variants, a subpopulation of S. aureus that is often defective in electron transport. Second-step mutants selected with NSFQ-105 had gyrA(S84L) alterations; those obtained with sparfloxacin carried a gyrA(D83A) mutation or a novel gyrB deletion (?RKSAL, residues 405 to 409) affecting a trypsin-sensitive region linking functional domains of S. aureus GyrB. Each mutation was associated with four- to eightfold increases in MICs of NSFQ-105 and sparfloxacin, but not of ciprofloxacin, which we confirm targets topoisomerase IV. The presence of wild-type grlB-grlA gene sequences in second-step mutants excluded involvement of topoisomerase IV in the small-colony phenotype. Growth revertants retaining mutant gyrA or gyrB alleles were quinolone susceptible, indicating that resistance to NSFQ-105 and sparfloxacin was contingent on the small-colony mutation. We propose that small-colony mutations unbalance target sensitivities, perhaps through altered ATP or topoisomerase levels, such that gyrase becomes the primary drug target. Breaking of target parity by genetic or physiological means eliminates the need for two target mutations and provides a novel mechanism for stepwise selection of quinolone resistance. PMID:12121924

  7. APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability.

    PubMed

    Lan, Xiqian; Jhaveri, Aakash; Cheng, Kang; Wen, Hongxiu; Saleem, Moin A; Mathieson, Peter W; Mikulak, Joanna; Aviram, Sharon; Malhotra, Ashwani; Skorecki, Karl; Singhal, Pravin C

    2014-08-01

    Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-?, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent. PMID:24899058

  8. Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa

    PubMed Central

    Swanson, Richard; Petitou, Maurice

    2012-01-01

    EP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (?-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (kon > 107M?1s?1), a high affinity (KI = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (KD = 30nM) and activated the serpin to rapidly (kass ? 106M?1s?1) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (? 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone. PMID:22144183

  9. Anti-HIV agents that selectively target retroviral nucleocapsid protein zinc fingers without affecting cellular zinc finger proteins.

    PubMed

    Huang, M; Maynard, A; Turpin, J A; Graham, L; Janini, G M; Covell, D G; Rice, W G

    1998-04-23

    Agents that target the two highly conserved Zn fingers of the human immunodeficiency virus (HIV) nucleocapsid p7 (NCp7) protein are under development as antivirals. These agents covalently modify Zn-coordinating cysteine thiolates of the fingers, causing Zn ejection, loss of native protein structure and nucleic acid binding capacity, and disruption of virus replication. Concentrations of three antiviral agents that promoted in vitro Zn ejection from NCp7 and inhibited HIV replication did not impact the functions of cellular Zn finger proteins, including poly(ADP-ribose) polymerase and the Sp1 and GATA-1 transcription factors, nor did the compounds inhibit HeLa nuclear extract mediated transcription. Selectivity of interactions of these agents with NCp7 was supported by molecular modeling analysis which (1) identified a common saddle-shaped nucleophilic region on the surfaces of both NCp7 Zn fingers, (2) indicated a strong correspondence between computationally docked positions for the agents tested and overlap of frontier orbitals within the nucleophilic loci of the NCp7 Zn fingers, and (3) revealed selective steric exclusion of the agents from the core of the GATA-1 Zn finger. Further modeling analysis suggests that the thiolate of Cys49 in the carboxy-terminal finger is the site most susceptible to electrophilic attack. These data provide the first experimental evidence and rationale for antiviral agents that selectively target retroviral nucleocapsid protein Zn fingers. PMID:9554870

  10. Selective inhibition of regulatory T cells by targeting the PI3K-Akt pathway.

    PubMed

    Abu-Eid, Rasha; Samara, Raed N; Ozbun, Laurent; Abdalla, Maher Y; Berzofsky, Jay A; Friedman, Kevin M; Mkrtichyan, Mikayel; Khleif, Samir N

    2014-11-01

    Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses are still needed. The temporary nature of the therapy-induced immune response can be attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, regulatory T cells (Treg). Although the depletion of Tregs has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. We found that PI3K-Akt pathway inhibitors selectively inhibit Tregs with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Tregs in comparison with Tconvs when treated with different Akt and PI3K inhibitors. This effect has been observed in both human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Tregs in both naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors led to a significant therapeutic antitumor effect, which was shown to be Treg dependent. Here, we report the use of PI3K-Akt pathway inhibitors as potent agents for the selective depletion of suppressive Tregs. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg depletion. PMID:25080445

  11. Genomic Expression Analyses Reveal Lysosomal, Innate Immunity Proteins, as Disease Correlates in Murine Models of a Lysosomal Storage Disorder

    PubMed Central

    Alam, Md. Suhail; Getz, Michelle; Safeukui, Innocent; Yi, Sue; Tamez, Pamela; Shin, Jenny; Velázquez, Peter; Haldar, Kasturi

    2012-01-01

    Niemann-Pick Type C (NPC) disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of Balb/c Npc1?/? mice relative to Npc1+/? at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher’s disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of Npc1?/? as well as Balb/c Npc1nmf164 mice (bearing a point mutation closer to human disease mutants) and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in Npc1?/? mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the Npc1?/? spleen and liver (where large foci were detected proximal to damaged tissue). Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs (hitherto not associated with NPC) suggests their role in pathophysiology and disease exacerbation. PMID:23094108

  12. Animal models for medications development targeting alcohol abuse using selectively bred rat lines: neurobiological and pharmacological validity.

    PubMed

    Bell, Richard L; Sable, Helen J K; Colombo, Giancarlo; Hyytia, Petri; Rodd, Zachary A; Lumeng, Lawrence

    2012-11-01

    The purpose of this review paper is to present evidence that rat animal models of alcoholism provide an ideal platform for developing and screening medications that target alcohol abuse and dependence. The focus is on the 5 oldest international rat lines that have been selectively bred for a high alcohol-consumption phenotype. The behavioral and neurochemical phenotypes of these rat lines are reviewed and placed in the context of the clinical literature. The paper presents behavioral models for assessing the efficacy of pharmaceuticals for the treatment of alcohol abuse and dependence in rodents, with particular emphasis on rats. Drugs that have been tested for their effectiveness in reducing alcohol/ethanol consumption and/or self-administration by these rat lines and their putative site of action are summarized. The paper also presents some current and future directions for developing pharmacological treatments targeting alcohol abuse and dependence. PMID:22841890

  13. Animal models for medications development targeting alcohol abuse using selectively bred rat lines: Neurobiological and pharmacological validity

    PubMed Central

    Bell, Richard L.; Sable, Helen J.K.; Colombo, Giancarlo; Hyytia, Petri; Rodd, Zachary A.; Lumeng, Lawrence

    2012-01-01

    The purpose of this review paper is to present evidence that rat animal models of alcoholism provide an ideal platform for developing and screening medications that target alcohol abuse and dependence. The focus is on the 5 oldest international rat lines that have been selectively bred for a high alcohol-consumption phenotype. The behavioral and neurochemical phenotypes of these rat lines are reviewed and placed in the context of the clinical literature. The paper presents behavioral models for assessing the efficacy of pharmaceuticals for the treatment of alcohol abuse and dependence in rodents, with particular emphasis on rats. Drugs that have been tested for their effectiveness in reducing alcohol/ethanol consumption and/or self-administration by these rat lines and their putative site of action are summarized. The paper also presents some current and future directions for developing pharmacological treatments targeting alcohol abuse and dependence. PMID:22841890

  14. Targeting the cannabinoid CB2 receptor: modelling and structural determinants of CB2 selective ligands

    PubMed Central

    Poso, A; Huffman, J W

    2007-01-01

    Recent developments indicate that CB2 receptor ligands have the potential to become therapeutically important. To explore this potential, it is necessary to develop compounds with high affinity for the CB2 receptor and little affinity for the CB1 receptor. This review will discuss structure-activity relations at both receptors for classical cannabinoids and cannabimimetic indoles. Examples of CB2 selective ligands from both classes of compounds are presented and the structural features leading to selectivity are described. Two approaches, receptor mutations and molecular modelling, have been employed to investigate the interaction of ligands with both cannabinoid receptors. These results obtained from these techniques are discussed. PMID:17982473

  15. Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs

    Microsoft Academic Search

    Déborah Tribouillard-Tanvier; Suzana Dos Reis; Fabienne Gug; Cécile Voisset; Vincent Béringue; Raimon Sabate; Ema Kikovska; Nicolas Talarek; Stéphane Bach; Chenhui Huang; Nathalie Desban; Sven J. Saupe; Surachai Supattapone; Jean-Yves Thuret; Stéphane Chédin; Didier Vilette; Hervé Galons; Suparna Sanyal; Marc Blondel

    2008-01-01

    Background: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology\\/Principal Findings: Here we report the identification of cellular targets of

  16. 768. Redirected T Cells to Selectively Target Mature B Cell Derived Malignancies

    Microsoft Academic Search

    Juan Vera; Barbara Savoldo; Stephane Vigouroux; Ettore Biagi; Martin Pule; Claudia Rossig; Malcolm Brenner; Gianpietro Dotti

    2006-01-01

    Adoptive transfer of CD19 or CD20 chimeric artificial receptors (CARs) to T-lymphocytes has been proposed to treat B-cell malignancies. If successful, however, the approach would likely severely impair humoral immunity. Since low grade lymphomas express surface monoclonal immunoglobulins (Igs) carrying kappa (?) or lambda (?) light chain, we explored whether chimeric T-cells targeting these immunoglobulin chains could be used instead.

  17. Selective apoptosis of breast cancer cells by siRNA targeting of BORIS

    Microsoft Academic Search

    Christopher J. Dougherty; Thomas E. Ichim; Liping Liu; Gary Reznik; Wei-Ping Min; Anahit Ghochikyan; Michael G. Agadjanyan; Boris N. Reznik

    2008-01-01

    Brother of the regulator of imprinted sites (BORIS) is an epigenetically acting transcription factor which represses the tumor inhibitor functions of the tumor suppressor protein CTCF. BORIS expression has not been documented in adult females, making it an exciting molecular target for drug development in breast cancer. Previously, we demonstrated that vaccination of mice with zing-finger (ZF)-deleted non-functional BORIS results

  18. Bioconjugation of Calcium Phosphate Nanoparticles for Selective Targeting of Human Breast and Pancreatic Cancers In Vivo

    PubMed Central

    Sharma, Rahul; Barth, Brian M.; Altino?lu, Erhan ?.; Morgan, Thomas T.; Shanmugavelandy, Sriram S.; Kaiser, James M.; McGovern, Christopher; Matters, Gail L.; Smith, Jill P.; Kester, Mark; Adair, James H.

    2010-01-01

    The early diagnosis of cancer is the critical element in successful treatment and long term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a ~20nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near infra-red imaging agent indocyanine green (ICG) is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin- or a novel PEG-maleimide-coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are over-expressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting and imaging of breast and pancreatic cancer tumors. PMID:20180585

  19. Selective Targeting of p53 Gene Mutational Hotspots in Human Cancers by Etiologically Defined Carcinogens

    Microsoft Academic Search

    Alain Puisieux; Susan Lim; John Groopman; Mehmet Ozturk

    In lung and liver cancers. pS3 mutations are mostly G:C to T:A transversions. This type of mutation is known to be induced by benzo(a)pyrcne and aflatoxin B, which are associated with the etiology of lung and liver cancers, respectively. Using a novel assay based on DNA polymerase fingerprint analysis, we identified p53 nucleotides targeted by these carcinogens. Thirteen of 14

  20. Selective Serotonin Reuptake Inhibitor Fluoxetine Inhibits Replication of Human Enteroviruses B and D by Targeting Viral Protein 2C

    PubMed Central

    Ulferts, Rachel; van der Linden, Lonneke; Thibaut, Hendrik Jan; Lanke, Kjerstin H. W.; Leyssen, Pieter; Coutard, Bruno; De Palma, Armando M.; Canard, Bruno; Neyts, Johan

    2013-01-01

    Although the genus Enterovirus contains many important human pathogens, there is no licensed drug for either the treatment or the prophylaxis of enterovirus infections. We report that fluoxetine (Prozac)—a selective serotonin reuptake inhibitor—inhibits the replication of human enterovirus B (HEV-B) and HEV-D but does not affect the replication of HEV-A and HEV-C or human rhinovirus A or B. We show that fluoxetine interferes with viral RNA replication, and we identified viral protein 2C as the target of this compound. PMID:23335743

  1. Synthesis of length-controlled polyvalent silver nanowire-DNA conjugates for sensitive and selective detection of DNA targets.

    PubMed

    Han, Sang Hun; Lee, Jae-Seung

    2012-01-10

    We have developed a facile method to rapidly synthesize the monodisperse silver nanowire-DNA conjugates with a constant diameter and systematically controllable lengths in the range of 0.5-2.5 ?m. The synthesis of silver nanowires takes advantage of poly(sodium 4-styrenesulfonate) as a structure-directing reagent and is performed under very mild conditions such as room temperature and aqueous media. The nanowires are densely conjugated with DNA sequences enough to exhibit the cooperative properties for the sensitive and selective detection of DNA targets. The limit of detection is 50 pM. PMID:22081915

  2. 6-(Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective inhibitors targeting hepatitis C virus NS4B.

    PubMed

    Chen, Guangming; Ren, Hongyu; Zhang, Nanjing; Lennox, William; Turpoff, Anthony; Paget, Steven; Li, Chunshi; Almstead, Neil; Njoroge, F George; Gu, Zhengxian; Graci, Jason; Jung, Stephen P; Colacino, Joseph; Lahser, Fred; Zhao, Xin; Weetall, Marla; Nomeir, Amin; Karp, Gary M

    2015-02-15

    A structure-activity relationship investigation of various 6-(azaindol-2-yl)pyridine-3-sulfonamides using the HCV replicon cell culture assay led to the identification of a potent series of 7-azaindoles that target the hepatitis C virus NS4B. Compound 2ac, identified via further optimization of the series, has excellent potency against the HCV 1b replicon with an EC50 of 2nM and a selectivity index of >5000 with respect to cellular GAPDH RNA. Compound 2ac also has excellent oral plasma exposure levels in rats, dogs and monkeys and has a favorable liver to plasma distribution profile in rats. PMID:25613678

  3. Rapid and Targeted Introgression of Genes into Popular Cultivars Using Marker-Assisted Background Selection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have optimized a marker-assisted background selection (MABS)-based gene introgression approach in wheat where 97% or more of a recurrent parent genome can be recovered in just two backcrosses (BCs). A four-step MABS method was developed based on ‘Plabsim’ computer simulations and wheat genome str...

  4. Spectroscopic Target Selection in the Sloan Digital Sky Survey: The Main Galaxy Sample

    Microsoft Academic Search

    David H. Weinberg; Robert H. Lupton; Vijay K. Narayanan; James Annis; Mariangela Bernardi; Michael Blanton; Scott Burles; A. J. Connolly; Julianne Dalcanton; Mamoru Doi; Daniel Eisenstein; Joshua A. Frieman; Masataka Fukugita; James E. Gunn; Zeljko Ivezic; Stephen Kent; Rita S. J. Kim; G. R. Knapp; Richard G. Kron; Heidi Jo Newberg; Sadanori Okamura; Thomas R. Quinn; Michael W. Richmond; David J. Schlegel; Kazuhiro Shimasaku; Mark SubbaRao; Alexander S. Szalay; Dan Vanden Berk; Michael S. Vogeley; Brian Yanny; Naoki Yasuda; Donald G. York; Idit Zehavi

    2002-01-01

    We describe the algorithm that selects the main sample of galaxies for spectroscopy in the Sloan Digital Sky Survey (SDSS) from the photometric data obtained by the imaging survey. Galaxy photometric properties are measured using the Petrosian magnitude system, which measures flux in apertures determined by the shape of the surface brightness profile. The metric aperture used is essentially independent

  5. Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy

    Microsoft Academic Search

    F. M. Platt; M. Jeyakumar; U. Andersson; D. A. Priestman; R. A. Dwek; T. D. Butters; T. M. Cox; R. H. Lachmann; C. E. M. Hollak; J. M. F. G. Aerts; S. Van Weely; M. Hrebícek; C. Moyses; I. Gow; D. Elstein; A. Zimran

    2001-01-01

    The glycosphingolipid (GSL) lysosomal storage diseases are caused by mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes. In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and disease. The diseases frequently have a progressive neurodegenerative course. The therapeutic options for treating these diseases are

  6. Lysosomal Membrane Permeabilization Induces Cell Death in a Mitochondrion-dependent Fashion

    Microsoft Academic Search

    Patricia Boya; Karine Andreau; Delphine Poncet; Naoufal Zamzami; Jean-Luc Perfettini; Didier Metivier; David M. Ojcius; Marja Jäättelä; Guido Kroemer

    2003-01-01

    A number of diseases are due to lysosomal destabilization, which results in damaging cell loss. To investigate the mechanisms of lysosomal cell death, we characterized the cytotoxic action of two widely used quinolone antibiotics: ciprofloxacin (CPX) or norfloxacin (NFX). CPX or NFX plus UV light (NFX * ) induce lysosomal membrane permeabilization (LMP), as detected by the release of cathepsins

  7. High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening

    E-print Network

    Gelb, Michael

    High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening Zdenek Spacil,1 Haribabu: There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the avail- ability spots from 58 random newborns and lysosomal storage disease­affected patients showed that the assay

  8. Protective effect of enterosgel on rat liver lysosomes during cytostatic treatment.

    PubMed

    Grek, O R; Mishenina, S V; Pupyshev, A B

    2002-10-01

    Polychemotherapy with a complex of cytostatics (cyclophosphamide, doxorubicin, vincristine, prednisolone) induces progressive damage to hepatocyte membranes, which manifested in labilization of lysosomes and activation of lysosomal enzymes and serum transaminases. Enterosgel stabilized liver lysosomes and reduced manifestation of hepatocyte cytolysis. PMID:12533758

  9. Aggregated LDL and lipid dispersions induce lysosomal cholesteryl ester accumulation in macrophage foam cells

    Microsoft Academic Search

    Evelyn E. Griffin; Jody C. Ullery; Brian E. Cox; W. Gray Jerome

    2005-01-01

    Macrophage foam cells in atherosclerotic lesions accumulate substantial cholesterol stores within large, swollen lysosomes. Previous studies with mildly oxidized low den- sity lipoprotein (OxLDL)-treated THP-1 macrophages sug- gest an initial buildup of free cholesterol (FC), followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis and a subsequent lysosomal accumulation of unhydrolyzed lipoprotein CE. We examined whether other potential sources

  10. New Roles for Lysosomal Trafficking in Morphogen Gradient Sensing

    NSDL National Science Digital Library

    Elena Rainero (Beatson Institute for Cancer Research; Integrin Cell Biology Laboratory REV)

    2011-05-03

    The way in which cells recognize their position in a gradient of morphogen controls differentiation during embryogenesis. New findings indicate that the rate at which internalized morphogen receptors are trafficked to lysosomes is key to the accurate and precise sensing of morphogen gradients and the appropriate initiation of differentiation programs during development.

  11. Neuronopathic Lysosomal Storage Diseases: Clinical and Pathologic Findings

    ERIC Educational Resources Information Center

    Prada, Carlos E.; Grabowski, Gregory A.

    2013-01-01

    Background: The lysosomal--autophagocytic system diseases (LASDs) affect multiple body systems including the central nervous system (CNS). The progressive CNS pathology has its onset at different ages, leading to neurodegeneration and early death. Methods: Literature review provided insight into the current clinical neurological findings,…

  12. Lysosomal storage diseases: Natural history and ethical and economic aspects

    Microsoft Academic Search

    Ernest Beutler

    2006-01-01

    Potential treatment for lysosomal diseases now includes enzyme replacement therapy, substrate reduction therapy, and chaperone therapy. The first two of these have been implemented commercially, and the spectrum of diseases that are now treatable has expanded from Gaucher disease to include several other disorders. Treatment of these diseases is extremely costly. We explore some of the reasons for the high

  13. Impaired trafficking of mutants of lysosomal glucocerebrosidase in Gaucher's disease

    Microsoft Academic Search

    Martina Schmitz; Marwan Alfalah; Johannes M. F. G. Aerts; Hassan Y. Naim; Klaus-Peter Zimmer

    2005-01-01

    Gaucher's disease is the most inherited lysosomal storage disorder. Except for a few cases, the broad phenotypic heterogeneity of Gaucher's disease can be neither predicted from defined mutations nor from differences in residual enzyme activity. Here, we analyse the intracellular trafficking of glucocerebrosidase as an underlying mechanism for the expression of the clinical phenotype. Biosynthetic labeling studies combined with immunofluorescence

  14. Tandem Mass Spectrometry for the Direct Assay of Lysosomal

    E-print Network

    Gelb, Michael

    is expected to be necessary to maximize the benefits of therapy. Therefore, we developed an assay for N-acetylgalactosamine a deficiency of lysosomal N-acetylgalactosamine-6-sulfate sulfatase (GALNS) (EC 3.1.6.4) activity (1). The enzyme hydrolyzes the sulfate ester bond of galactose 6-sulfate in keratan sul- fate and of N-acetylgalactosamine

  15. Mitochondrial Ca2+ homeostasis in lysosomal storage diseases.

    PubMed

    Kiselyov, Kirill; Muallem, Shmuel

    2008-07-01

    Lysosomal storage diseases (LSDs) are a class of genetic disorders in which proteins responsible for digestion or absorption of endocytosed material do not function or do not localize properly. The resulting cellular "indigestion" causes buildup of intracellular storage inclusions that contain unprocessed lipids and proteins that form macromolecular complexes. The buildup of storage material is associated with degenerative processes that are observed in all LSDs, albeit the correlation between the amount of storage inclusions and the severity of the degenerative processes is not always evident. The latter suggests that a specific mechanism set in motion by aberrant lysosomal function drives the degenerative processes in LSDs. It is becoming increasingly clear that in addition to their function in degrading endocytosed material, lysosomes are essential housekeeping organelles responsible for maintaining healthy population of intracellular organelles, in particular mitochondria. The present review surveys the current knowledge on the lysosomal-mitochondrial axis and its possible role as a contributing factor to mitochondrial Ca(2+) homeostasis and to cell death in LSDs. PMID:18242695

  16. Genetic Regulation of Caenorhabditis elegans Lysosome Related Organelle Function

    PubMed Central

    Soukas, Alexander A.; Carr, Christopher E.; Ruvkun, Gary

    2013-01-01

    Lysosomes are membrane-bound organelles that contain acid hydrolases that degrade cellular proteins, lipids, nucleic acids, and oligosaccharides, and are important for cellular maintenance and protection against age-related decline. Lysosome related organelles (LROs) are specialized lysosomes found in organisms from humans to worms, and share many of the features of classic lysosomes. Defective LROs are associated with human immune disorders and neurological disease. Caenorhabditis elegans LROs are the site of concentration of vital dyes such as Nile red as well as age-associated autofluorescence. Even though certain short-lived mutants have high LRO Nile red and high autofluorescence, and other long-lived mutants have low LRO Nile red and low autofluorescence, these two biologies are distinct. We identified a genetic pathway that modulates aging-related LRO phenotypes via serotonin signaling and the gene kat-1, which encodes a mitochondrial ketothiolase. Regulation of LRO phenotypes by serotonin and kat-1 in turn depend on the proton-coupled, transmembrane transporter SKAT-1. skat-1 loss of function mutations strongly suppress the high LRO Nile red accumulation phenotype of kat-1 mutation. Using a systems approach, we further analyzed the role of 571 genes in LRO biology. These results highlight a gene network that modulates LRO biology in a manner dependent upon the conserved protein kinase TOR complex 2. The results implicate new genetic pathways involved in LRO biology, aging related physiology, and potentially human diseases of the LRO. PMID:24204312

  17. Edinburgh Research Explorer Pneumolysin Activates Macrophage Lysosomal Membrane

    E-print Network

    Millar, Andrew J.

    Lysosomal2014. Martin A. Bewley, Michael Naughton, Julie Preston, et al. Formation Mechanisms without and Executes Apoptosis by Distinct Mechanisms without Membrane Pore Formation Martin A. Bewley,a,b Michael Naughton,a Julie Preston,a Andrea Mitchell,d Ashleigh Holmes,e Helen M. Marriott,a,b Robert C. Read

  18. Unexpected regulation of a `housekeeping gene:` Spatial and temporal expression of a multifunctional lysosomal gene, prosaposin

    SciTech Connect

    Sun, Y.; Witte, D.; Grabowski, G.A. [Children`s Hospital Research Foundation, Cincinnati, OH (United States)

    1994-09-01

    Prosaposin is a multifunctional locus in man and mice which encodes a tandem and in the same reading frame four glycoprotein activators or saposins of lysosomal hydrolases. These ubiquitously expressed glycoproteins and the precursor, prosaposin, have been proposed to function in glycosphingolipid catabolic pathways and glycolipid transport. Like other lysosomal genes, it was thought to be regulated at a low level, a `housekeeping gene`; this was not the case. To characterize the temporal and spatial expression of the prosaposin loci, prenatal and postnatal mouse tissues were screened with the murine antisense riboprobe by in situ hybridization. Prenatally, prosaposin mRNA was expressed differentially in decidual layers of the placenta, particularly, the decidua capsularis. The dorsal root ganglia also showed higher levels expression than surrounding tissues. In comparison, high level differential expression of prosaposin was clearly evident postnatally in a variety of organs. In secretory epithelial cells of the choroid plexus, ventricular lining, upper trachea, esophagus, cortical renal tubules of the kidney, epididymis and sertoli cells of the testes. Discrete localization of prosaposin mRNA expression was also found in neurons of the cerebral cortex, cerebellar cortex and lateral columns of the spinal cord as well as in hepatocytes of the mature liver. Very high levels of expression were found in lipid-secreting cells of the Harderian glands and corpus luteum of the ovary, macrophages of intestinal lymph nodes, splenic tissue and thymus. Studies in selected human tissues indicate a similar pattern of expression. These studies show specific spatial and temporal prosaposin expression in a heretofore thought of lysosomal `housekeeping` locus that suggests a pivitol role for this locus in the tissue expression of glycosphingolipid storage diseases.

  19. Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis

    PubMed Central

    1982-01-01

    Certain species of Chlorella live within the digestive cells of the fresh water cnidarian Hydra viridis. When introduced into the hydra gut, these symbiotic algae are phagocytized by digestive cells but avoid host digestion and persist at relatively constant numbers within host cells. In contrast, heat-killed symbionts are rapidly degraded after phagocytosis. Live symbionts appear to persist because host lysosomes fail to fuse with phagosomes containing live symbionts. Neither acid phosphatase nor ferritin was delivered via lysosomes into phagosomes containing live symbionts, whereas these lysosomal markers were found in 50% of the vacuoles containing heat-killed symbionts 1 h after phagocytosis. Treatment of symbiotic algae before phagocytosis with polycationic polypeptides abolishes algal persistence and perturbs the ability of these algae to control the release of photosynthate in vitro. Similarly, inhibition of photosynthesis and hence of the release of photosynthetic products as a result of prolonged darkness and 3-(3,4- dichlorophenyl)-1,1-dimethyl urea (DCMU) treatment also abolishes persistence. Symbiotic algae are not only protected from host digestive attack but are also selectively transported within host cells, moving from the apical site of phagocytosis to a basal position of permanent residence. This process too is disrupted by polycationic polypeptides, DCMU and darkness. Both algal persistence and transport may, therefore, be a function of the release of products from living, photosynthesizing symbionts. Vinblastine treatment of host animals blocked the movement of algae within host cells but did not perturb algal persistence: algal persistence and the transport of algae may be initiated by the same signal, but they are not interdependent processes. PMID:7119017

  20. Highly sensitive and selective detection of miRNA: DNase I-assisted target recycling using DNA probes protected by polydopamine nanospheres.

    PubMed

    Xie, Yi; Lin, Xiaoyan; Huang, Yishun; Pan, Rujun; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong James

    2015-02-01

    Based on the protective properties of polydopamine nanospheres for DNA probes against nuclease digestion, we have developed a DNase I-assisted target recycling signal amplification method for highly sensitive and selective detection of miRNA. PMID:25554948

  1. Current insights into the pathophysiology of Alzheimer's disease: selecting targets for early therapeutic intervention.

    PubMed

    Hampel, Harald

    2012-08-01

    The development of therapies for Alzheimer's disease (AD) presents numerous challenges for physicians, researchers, and the pharmaceutical industry, with many drug candidates showing promise at one stage of clinical research only to fall at the next hurdle. A great number of drugs with a variety of targets and clusters of mechanisms are currently in various stages of basic and clinical investigation. However, these hypothesis-derived agents may be tested much too late in the chronically progressive disease process to demonstrate meaningful effects or outcomes, mirroring the clinical syndromal scenario in which the underlying pathophysiological disease condition is frequently diagnosed extremely late. Moreover, the complexity of the disease calls for developments and improvements in study designs and methods modeled for different target populations and disease stages (e.g. asymptomatic to prodromal to syndromal). New integrated concepts and models of disease pathophysiology, use of validated and qualified biomarkers, outcomes and endpoints, particularly the development of a surrogate outcome, may allow targeting of characteristic mechanism-derived therapies of specifically affected biological systems at different time-points in the disease process, providing increasing opportunities for early and preventative intervention. A core set of feasible diagnostic and predictive biomarkers is already validated and in the process of standardization; however, continued and intensified research efforts will likely reveal a variety of novel biomarkers that grasp the complexity of the underlying disease process. In the future, trials of drugs to modify and prevent AD may embrace enrichment strategies and maybe be stratified by disease stage, genetic factors as well as by disease endophenotypes. PMID:22784422

  2. Selection and calculation of overall parameters of laser communicator applied for vehicle mobile target

    NASA Astrophysics Data System (ADS)

    Zhang, Hongtao; Yin, FuChang; Shi, Jiangtian

    2002-09-01

    The model of laser communication system using in vehicle mobile target was built. Optimal design and calculation was analyzed for system performance, such as laser transmission, photoelectric reception and speech information processing and image stabilization. Algorithm of MELP (Mixed Excitation Line Predication) compressed speech scrambling was received in processing voice message, which reached 2.4 kbps digital speech code stream, and realized digitization of laser communication. It was also analyzed carefully about the principle theory of MELP algorithm encoding and decoding as well as algorithm realization used in DSP. Image stabilization and tracking were realized by process signal errors which acquired at quadrant error sensors.

  3. 6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site

    PubMed Central

    Ghizzoni, Massimo; Wu, Jiang; Gao, Tielong; Haisma, Hidde J.; Dekker, Frank J.; Zheng, Y. George

    2011-01-01

    Histone acetyltransferases are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elucidation of the biological functions of HATs and may also have pharmacological value for potential new therapies. In this work, analogs of the known HAT inhibitor anacardic acid were synthesized and evaluated for inhibition of HAT activity. Biochemical assays revealed novel anacardic acid analogs that inhibited the human recombinant enzyme Tip60 selectively compared to PCAF and p300. Enzyme kinetics studies demonstrated that inhibition of Tip60 by one such novel anacardic acid derive, 20, was essentially competitive with Ac-CoA and noncompetitive with the histone substrate. In addition, these HAT inhibitors effectively inhibited acetyltransferase activity of nuclear extracts on the histone H3 and H4 at micromolar concentrations. PMID:22100137

  4. Leaky lysosomes in lung transplant macrophages: azithromycin prevents oxidative damage

    PubMed Central

    2012-01-01

    Background Lung allografts contain large amounts of iron (Fe), which inside lung macrophages may promote oxidative lysosomal membrane permeabilization (LMP), cell death and inflammation. The macrolide antibiotic azithromycin (AZM) accumulates 1000-fold inside the acidic lysosomes and may interfere with the lysosomal pool of Fe. Objective Oxidative lysosomal leakage was assessed in lung macrophages from lung transplant recipients without or with AZM treatment and from healthy subjects. The efficiency of AZM to protect lysosomes and cells against oxidants was further assessed employing murine J774 macrophages. Methods Macrophages harvested from 8 transplant recipients (5 without and 3 with ongoing AZM treatment) and 7 healthy subjects, and J774 cells pre-treated with AZM, a high-molecular-weight derivative of the Fe chelator desferrioxamine or ammonium chloride were oxidatively stressed. LMP, cell death, Fe, reduced glutathione (GSH) and H-ferritin were assessed. Results Oxidant challenged macrophages from transplants recipients without AZM exhibited significantly more LMP and cell death than macrophages from healthy subjects. Those macrophages contained significantly more Fe, while GSH and H-ferritin did not differ significantly. Although macrophages from transplant recipients treated with AZM contained both significantly more Fe and less GSH, which would sensitize cells to oxidants, these macrophages resisted oxidant challenge well. The preventive effect of AZM on oxidative LMP and J774 cell death was 60 to 300 times greater than the other drugs tested. Conclusions AZM makes lung transplant macrophages and their lysososomes more resistant to oxidant challenge. Possibly, prevention of obliterative bronchiolitis in lung transplants by AZM is partly due to this action. PMID:23006592

  5. Selection of relevant non-target herbivores for monitoring the environmental effects of Bt maize pollen.

    PubMed

    Schmitz, Gregor; Bartsch, Detlef; Pretscher, Peter

    2003-01-01

    Genes of Bacillus thuringiensis var. kurstaki (Berliner) that encode lepidopteran-specific toxins were engineered into maize for protection against the European Corn Borer, Ostrinia nubilalis (Hbn.). Recent data suggest that Lepidoptera may be negatively affected, if maize pollen contains high amounts of Bt toxin and is diposited on host plants near maize fields. Monitoring the environmental effects of commercial Bt maize fields requires effective use of limited financial and logistical resources. The aim of this study was to develop and apply tools for selecting relevant herbivore species for the field monitoring of environmental Bt toxin effects via pollen deposition. We first present a theoretical selection tree based on "risk index of Bt pollen for herbivores" (I(Btp)). Our index consists of five classes from zero (not relevant) to four (highly relevant) derived from data on potential temporal and spatial coincidence of pollen exposure (A), feeding mode (B), susceptibility to lepidopteran-specific Bt toxins (C) and hazard to rare and/or endangered species ("Red List") (D). We then screened the Macrolepidoptera database LEPIDAT to identify relevant species in Germany. Finally, we also applied the index to species found in a local biocoenotic field study (Bonn, Western Rhineland, Germany). Approximately 7% of the German Macrolepidoptera species mainly occur in farmland areas and were selected as being potentially affected by Bt pollen exposure. Of these species, 14% (= 1% of total) were found to be potentially exposed on a regional scale. The combination of I(Btp) and database screening enables us to pre-select species for monitoring purposes. PMID:15612277

  6. Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity

    PubMed Central

    Almaguel, Frankis G.; Liu, Jo-Wen; Pacheco, Fabio J.; De Leon, Daisy; Casiano, Carlos A.; De Leon, Marino

    2010-01-01

    Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity. PMID:20043885

  7. Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity

    PubMed Central

    Qiao, Liyan; Hamamichi, Shusei; Caldwell, Kim A; Caldwell, Guy A; Yacoubian, Talene A; Wilson, Scott; Xie, Zuo-Lei; Speake, Lisa D; Parks, Rachael; Crabtree, Donna; Liang, Qiuli; Crimmins, Stephen; Schneider, Lonnie; Uchiyama, Yasuo; Iwatsubo, Takeshi; Zhou, Yi; Peng, Lisheng; Lu, YouMing; Standaert, David G; Walls, Ken C; Shacka, John J; Roth, Kevin A; Zhang, Jianhua

    2008-01-01

    ?-synuclein (?-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. ?-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for ?-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous ?-syn in neurons without overabundance of ?-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for ?-syn metabolism. Conversely, CD overexpression reduces ?-syn aggregation and is neuroprotective against ?-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates ?-syn accumulation while its overexpression is protective against ?-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in ?-syn degradation and identify CD as a novel target for LB disease therapeutics. PMID:19021916

  8. Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

    PubMed Central

    Nemazanyy, Ivan; Blaauw, Bert; Paolini, Cecilia; Caillaud, Catherine; Protasi, Feliciano; Mueller, Amelie; Proikas-Cezanne, Tassula; Russell, Ryan C; Guan, Kun-Liang; Nishino, Ichizo; Sandri, Marco; Pende, Mario; Panasyuk, Ganna

    2013-01-01

    The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7?/? mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15?/? mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases. PMID:23630012

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