Science.gov

Sample records for selective lysosomal targeting

  1. 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

    investigated whether the defective lysosomal acidification associated with some cancer cells can be exploited to enhance selectivity of weakly basic anticancer agents. Normal cells typically have very acidic lysosomes, which provide a driving force...

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

    PubMed

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

    2015-07-01

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

  3. Two rhodamine lactam modulated lysosome-targetable fluorescence probes for sensitively and selectively monitoring subcellular organelle pH change.

    PubMed

    Li, Hongmei; Wang, Cuiling; She, Mengyao; Zhu, Yuelu; Zhang, Jidong; Yang, Zheng; Liu, Ping; Wang, Yaoyu; Li, Jianli

    2015-11-01

    Be a powerful technique for convenient detection of pH change in living cells, especially at subcellular level, fluorescent probes has attracted more and more attention. In this work, we designed and synthesized three rhodamine lactam modulated fluorescent probes RS1, RS2 and RS3, which all respond sensitively toward weak acidity (pH range 4-6) via the photophysical property in buffer solution without interference from the other metal ions, and they also show ideal pKa values and excellent reversibility. Particularly, by changing the lone pair electrons distribution of lactam-N atom with different conjugations, RS2 and RS3 exhibit high quantum yield, negligible cytotoxicity and excellent permeability. They are suitable to stain selectively lysosomes of tumor cells and monitor its pH changes sensitively via optical molecular imaging. The above findings suggest that the probes we designed could act as ideal and easy method for investigating the pivotal role of H(+) in lysosomes and are potential pH detectors in disease diagnosis through direct intracellular imaging. PMID:26572844

  4. Screening and Optimization of Ligand Conjugates for Lysosomal Targeting

    PubMed Central

    Meerovich, Igor; Koshkaryev, Alexander; Thekkedath, Ritesh; Torchilin, Vladimir P.

    2011-01-01

    The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide good rate of co-localization well the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared with plain liposomes. These results were additionally confirmed by the flow cytometry of the intact cells treated with liposomes loaded with with 5-dodecanoylaminofluorescein di-?-D-galactopyranoside, a specific substrate for the intralysosomal ?-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands – rhodamine B DSPE-PEG2k-amide and 6-(3-(DSPE-PEG2k)-thioureido) rhodamine B – demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the treatment of lysosome-associated disorders. PMID:21913714

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

    PubMed

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

    2015-07-01

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

  6. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2008-01-01

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

  8. Vesicular disruption of lysosomal targeting organometallic polyarginine bioconjugates.

    PubMed

    Gross, Annika; Alborzinia, Hamed; Piantavigna, Stefania; Martin, Lisandra L; Wölfl, Stefan; Metzler-Nolte, Nils

    2015-02-01

    Compounds which are able to destabilize the lysosomal membrane have been proposed as interesting candidates for targeted anticancer drugs due to the pronounced lysosomal changes in cancer cells. For this purpose, metallocene derivatives of a cell penetrating polyarginine peptide M–(Arg)9(Phe)2Lys–NH2 (where M = ferrocene carboxylate or ruthenocene carboxylate) were designed and their biological activities were investigated in detail. The ferrocenoyl- and ruthenocenoyl polyarginine bioconjugates were synthesized via Fmoc solid-phase peptide synthesis (SPPS) protocols on a microwave-assisted synthesizer. After HPLC purification >98% purity was observed for all conjugates. Their interaction with supported biomimetic membranes was investigated on a quartz crystal microbalance (QCM) and revealed a very strong binding of the metallocene peptides and their metal-free congeners to an artificial eukaryotic membrane model (DMPC–cholesterol). To demonstrate their antiproliferative utility as cytotoxic compounds for a targeted anticancer drug, cell viability (by the crystal violet assay), apoptosis (flow cytometry, Ann V/PI staining), induction of reactive oxygen species (ROS, by flow cytometry with dihydroethidium staining), and changes in cancer cell metabolism, e.g. respiration and glycolysis, were studied. Our results reveal only a weak toxicity for the metal-free polyarginine peptide, which could be significantly enhanced (to ca. 50 ?M against HeLa cells in the best case) by coupling ferrocene or ruthenocene carboxylates to the N-terminus of the peptide. The investigation of the cellular uptake and intracellular localization by fluorescence microscopy revealed an enhanced vesicular disruption by the metallocene bioconjugate compared to the metal-free derivative which could be triggered by light and chemicals. Further studies of apoptosis, respiration, glycolysis and ROS formation reveal the superior characteristics of the metallocene compounds. While most cells remain viable even at 300 ?M of the metal free bioconjugate 1, most cells are dead or in late stages of apoptosis at 200 ?M of the ruthenocene derivative 3, and at 100 ?M of the most active ferrocene derivative 2, however, all show very little sign of necrosis. Also, the metal free compound 1 does not induce ROS formation but both metallocene–polyarginine bioconjugates are clearly associated with enhanced intracellular ROS levels, with levels for the redox-active ferrocene derivative being two times higher than for the structurally very similar but redox-silent ruthenocene derivative. We propose that such metallocene–polyarginine peptides induce lysosomal membrane permeabilization and thereby could be developed towards targeted anticancer drugs. PMID:25608481

  9. High Resolution Crystal Structure of Human ?-Glucuronidase Reveals Structural Basis of Lysosome Targeting

    PubMed Central

    Hassan, Md. Imtaiyaz; Waheed, Abdul; Grubb, Jeffery H.; Klei, Herbert E.; Korolev, Sergey; Sly, William S.

    2013-01-01

    Human ?-glucuronidase (GUS) cleaves ?-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene. PMID:24260279

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

    PubMed

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

    2015-12-15

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

  11. 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

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

    PubMed Central

    Barriere, Herve; Nemes, Csilla; Du, Kai

    2007-01-01

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

  13. 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

  14. Lysosome-targeted octadecyl-rhodamine B-liposomes enhance lysosomal accumulation of glucocerebrosidase in Gaucher’s cells in vitro

    PubMed Central

    Thekkedath, Ritesh; Koshkaryev, Alexander; Torchilin, Vladimir P

    2013-01-01

    Aim We hypothesized that liposomes modified with lysosomotropic octadecyl-rhodamine B (Rh) and loaded with therapeutic glucocerebroside velaglucerase alfa (VPRIV™) will improve lysosomal delivery of the enzyme into Gaucher’s cells. Materials & methods Confocal microscopy and flow cytometry were used to evaluate the ability of Rh-modified liposomes loaded with VPRIV to improve the lysosomal targeting in monocyte-derived macrophages and Gaucher’s fibroblasts. Results Confocal microscopy demonstrated that Rh-modified liposomes localized primarily in the lysosomes. As confirmed by flow cytometry using specific substrate 5-(pentafluorobenzoylamino)fluorescein diglucoside, intralysosomal accumulation of VPRIV in the cells treated with Rh-modified liposomes was significantly increased (up to 68%) relative to the cells treated with plain liposomes or free VPRIV. Conclusion Rh-modified lysosomotropic liposomes can improve lysosomal accumulation of liposomal enzymes both in nonphagocytic Gaucher’s fibroblasts and phagocytic monocyte-derived macrophages. PMID:23199221

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

    SciTech Connect

    Haylett, T.; Thilo, L.

    1986-10-01

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

  16. 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

  17. 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

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

    PubMed

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

    2015-10-27

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

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

    PubMed

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

    2015-02-11

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

  20. Glycosylation-independent Lysosomal Targeting of Acid ?-Glucosidase Enhances Muscle Glycogen Clearance in Pompe Mice*

    PubMed Central

    Maga, John A.; Zhou, Jianghong; Kambampati, Ravi; Peng, Susan; Wang, Xu; Bohnsack, Richard N.; Thomm, Angela; Golata, Sarah; Tom, Peggy; Dahms, Nancy M.; Byrne, Barry J.; LeBowitz, Jonathan H.

    2013-01-01

    We have used a peptide-based targeting system to improve lysosomal delivery of acid ?-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients. PMID:23188827

  1. 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.

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

    PubMed Central

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

    2015-01-01

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

  3. 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

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

    PubMed

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

    2015-05-13

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

  5. Burglar Target Selection

    PubMed Central

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

    2015-01-01

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

  6. 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

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

    PubMed

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

    2015-12-24

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

  8. 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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    2015-11-01

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

  12. 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

  13. 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

  14. DRUG INDUCED PHOSPHOLIPIDOSIS: AN ACQUIRED LYSOSOMAL STORAGE DISORDER

    PubMed Central

    Shayman, James A.; Abe, Akira

    2012-01-01

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

  15. Sphingomyelins suppress the targeted disruption of lysosomes/endosomes by the photosensitizer NPe6 during photodynamic therapy

    PubMed Central

    2005-01-01

    Recent studies have described a biochemical pathway whereby lysosome disruption and the released proteases initiate the intrinsic apoptotic pathway. Irradiation of murine hepatoma 1c1c7 cells preloaded with the lysosomal photosensitizer NPe6 (N-aspartyl chlorin e6) caused a rapid loss of Acridine Orange staining of acidic organelles, release of cathepsin D from late endosomes/lysosomes and the activation of procaspase-3. Pretreatment of NPe6-loaded cultures with 10–50 ?M 3-O-MeSM (3-O-methylsphingomyelin) caused a concentration-dependent suppression of apoptosis following irradiation. This suppression reflected a stabilization of lysosomes/endosomes, as opposed to an inhibition of the accumulation of photosensitizer in these organelles. Exogenously added sphingomyelin, at comparable concentrations, offered some protection, but less than 3-O-MeSM. Fluorescence microscopy showed that 3-O-MeSM competed with NBD-C6-sphingomyelin (6-{[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl} sphingosyl phosphocholine) for co-localization with LysoTracker Red in acidic organelles. Pre-treatment of 1c1c7 cultures with 3-O-MeSM also suppressed the induction of apoptosis by TNF? (tumour necrosis factor ?), but offered no protection against HA14-1 [ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate], staurosporine, tunicamycin or thapsigargin. These results suggest that exogenously added 3-O-MeSM is trafficked to and stabilizes late endosomes/lysosomes against oxidant-induced damage, and further implicate a role for lysosomal proteases in the apoptotic processes initiated by TNF? and lysosomal photosensitizers. PMID:15943580

  16. Lysosomal Trafficking Regulator (LYST).

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-11-01

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

  1. Selective Targeting to Glioma with Nucleic Acid Aptamers

    PubMed Central

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

    2015-01-01

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

  2. 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.

  3. PIPing on lysosome tubes

    PubMed Central

    Ktistakis, Nicholas T; Tooze, Sharon A

    2013-01-01

    EMBO J (2013) 32, 324–339 doi:10.1038/emboj.2012.341; published online 12212012 The role of lysosomes in important cellular responses, including phagocytosis, cell surface repair, and autophagy underlies a number of human diseases. Furthermore, the role of the lysosomal surface in TORC1 signalling has revealed unexpected properties of these organelles. In this issue, Sridhar et al (2013) uncover an important role for PI(4)P for lysosome function under normal nutrient conditions and after prolonged nutrient deprivation. Ana Maria Cuervo, the late Dennis Shields, and colleagues (Sridhar et al, 2013) conclude that PI4 kinase III? on the surface of the lysosome controls the fidelity of sorting from the lysosome, and is required for autophagic lysosome reformation (ALR). These novel findings provide important insights into the complexities of the lipid composition of the lysosome, and how these lipids may control lysosome function. PMID:23314746

  4. Target Selection for the TESS Mission

    NASA Astrophysics Data System (ADS)

    Pepper, Joshua; Stassun, Keivan

    2015-12-01

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

  5. Agilent Technologies SureSelect Target

    E-print Network

    Kopp, Artyom

    Agilent Technologies SureSelect Target Enrichment System Illumina Paired-End Sequencing Platform and 2008 Illumina, Inc. All rights reserved. Only for use with the Illumina Genome Analyzer and associated

  6. Optogenetic acidification of synaptic vesicles and lysosomes.

    PubMed

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

    2015-12-01

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

  7. An Evaluation of Techniques for Selecting Moving Targets

    E-print Network

    compare our two techniques to unassisted selection in a controlled experiment. The results show that for moving target selection, Comet Tails and Target Lock can outperform unassisted selection, and result for assisted target selection with Comet Tails and Target Lock than unassisted selection. Keywords Moving

  8. 78 kDa receptor for Man6P-independent lysosomal enzyme targeting: Biosynthetic transport from endoplasmic reticulum to 'high-density vesicles'

    SciTech Connect

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

    2006-04-15

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

  9. Target selection for the HRIBF Project

    SciTech Connect

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

    1994-12-31

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

  10. Spectral Signatures of Saccade Target Selection.

    PubMed

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

    2016-01-01

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

  11. MaNGA: Target selection and Optimization

    NASA Astrophysics Data System (ADS)

    Wake, David

    2016-01-01

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

  12. MaNGA: Target selection and Optimization

    NASA Astrophysics Data System (ADS)

    Wake, David

    2015-01-01

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

  13. 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 ...

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

    PubMed Central

    Ciechanover, Aaron

    2012-01-01

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

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

    PubMed

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

    2015-12-15

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

  16. Methods for Probing Lysosomal Membrane Permeabilization.

    PubMed

    Jäättelä, Marja; Nylandsted, Jesper

    2015-01-01

    Cell death triggered by lysosomal membrane permeabilization (LMP) is gaining increased interest as target for cancer therapy, but the death pathway also plays an important role in normal physiology (e.g., during involution of the mammary gland). LMP-induced cell death is triggered by release of hydrolases including cysteine cathepsin proteases from the lysosomal lumen into the cytosol. Limited release of proteases to the cytoplasm induces apoptosis or apoptosis-like cell death, whereas massive LMP results in rapid cellular necrosis. Here we introduce three complementary methods for quantifying and visualizing LMP: (i) monitoring LMP by immunocytochemistry, (ii) visualizing LMP by fluorescent dextran release, and (iii) quantification of LMP by activity measurements of lysosomal enzymes in digitonin-extracted cytosol. PMID:26527770

  17. Lysosomal lipid storage diseases.

    PubMed

    Schulze, Heike; Sandhoff, Konrad

    2011-06-01

    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

  18. 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

  19. Transit Target Selection Using Reduced Proper Motions

    E-print Network

    Andrew Gould; Christopher W. Morgan

    2002-09-26

    In searches for planetary transits in the field, well over half of the survey stars are typically giants or other stars that are too large to permit straightforward detection of planets. For all-sky searches of bright V<~11 stars, the fraction is ~90%. We show that the great majority of these contaminants can be removed from the sample by analyzing their reduced proper motions (RPMs): giants have much lower RPMs than dwarfs of the same color. We use Hipparcos data to design a RPM selection function that eliminates most evolved stars, while rejecting only 9% of viable transit targets. Our method can be applied using existing or soon-to-be-released all-sky data to stars V<12.5 in the northern hemisphere and V<12 in the south. The method degrades at fainter magnitudes, but does so gracefully. For example, at V=14 it can still be used to eliminate giants redward of V-I~0.95, that is, the blue edge of the red giant clump.

  20. SureSelectXT Target Enrichment System for

    E-print Network

    Richardson, David

    SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Illumina Hi;SureSelectXT Target Enrichment System Kit for Illumina Multiplexed Sequencing Notices © Agilent understood and met. Acknowledgement Oligonucleotide sequences © 2006, 2008, and 2011 Illumina, Inc. All

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2011-01-01

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

  3. 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

  4. Target selection bias transfers across different response actions

    PubMed Central

    Moher, Jeff; Song, Joo-Hyun

    2014-01-01

    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. Pre-trial 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 following “no-go” trials where 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

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

    NASA Astrophysics Data System (ADS)

    Duncan, Gregg A.; Bevan, Michael A.

    2015-09-01

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

  6. Neuroinflammatory paradigms in lysosomal storage diseases

    PubMed Central

    Bosch, Megan E.; Kielian, Tammy

    2015-01-01

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

  7. 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

  8. 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

  9. Sexual selection targets cetacean pelvic bones

    PubMed Central

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

    2014-01-01

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

  10. 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

  11. Selectively targeting pain in the trigeminal system

    PubMed Central

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

    2015-01-01

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

  12. 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

  13. 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

  14. A SIMPLE LIKELIHOOD METHOD FOR QUASAR TARGET SELECTION

    SciTech Connect

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

    2011-12-20

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

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

    PubMed Central

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

    2011-01-01

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

  16. 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

  17. Mannose 6-phosphate-independent Lysosomal Sorting of LIMP-2.

    PubMed

    Blanz, Judith; Zunke, Friederike; Markmann, Sandra; Damme, Markus; Braulke, Thomas; Saftig, Paul; Schwake, Michael

    2015-10-01

    The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-phosphate (M6P)-independent trafficking receptor for ?-glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP-2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP-2 with the cation-independent M6P receptor (MPR) results in M6P-dependent targeting of LIMP-2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P-dependent delivery of LIMP-2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP-2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P-forming N-acetylglucosamine (GlcNAc)-1-phosphotransferase, LIMP-2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP-2 levels within lysosomes purified from liver of wild-type (wt) and GlcNAc-1-phosphotransferase-defective mice. Heterologous expression of the luminal domain of LIMP-2 in wild-type, LIMP-2-deficient and GlcNAc-1-phosphotransferase-defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP-2. Finally, cathepsin Z, a known GlcNAc-1-phosphotransferase substrate, but not LIMP-2, could be precipitated with M6P-specific antibodies. These data prove M6P-independent lysosomal sorting of LIMP-2 and subsequently GC in vivo. PMID:26219725

  18. 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.

  19. TARGET SELECTION FOR THE LBTI EXOZODI KEY SCIENCE PROGRAM

    SciTech Connect

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

    2015-02-01

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

  20. 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 ...

  1. Clearance of lysosomal glycogen accumulation by Transcription factor EB (TFEB) in muscle cells from lysosomal alpha-glucosidase deficient mice

    PubMed Central

    Li, Hoi Ming; Feeney, Erin; Li, Lishu; Zare, Hossein; Puertollano, Rosa; Raben, Nina

    2013-01-01

    A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in Glycogen Storage Disease type II (a severe metabolic myopathy and a paradigm for LSDs, also called Pompe disease) as the currently available therapy, replacement of the missing enzyme acid alpha-glucosidase, fails to reverse skeletal muscle pathology. Pompe disease is characterized by both lysosomal abnormality and dysfunctional autophagy. Here we show that TFEB is a viable therapeutic target in Pompe disease: overexpression of TFEB in a newly established conditionally immortalized skeletal muscle cell model reduced glycogen load and lysosomal size; and in the muscle fibers of GFP-LC3 Pompe disease mouse model significantly increased the motility of lysosomes in the fibers, and stimulated the fusion between lysosomes and autophagosomes under stress. Hence, modulation of TFEB activity holds promise for the development of a better therapy. In addition, the newly developed mouse and cell models have many potential applications such as large-scale drug screening for Pompe disease. PMID:23416076

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

    PubMed Central

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

    2008-01-01

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

  3. Feature Extraction and Selection Strategies for Automated Target Recognition

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

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

  4. Target selection and determination of function in structural genomics.

    PubMed

    Watson, James D; Todd, Annabel E; Bray, James; Laskowski, Roman A; Edwards, Aled; Joachimiak, Andrzej; Orengo, Christine A; Thornton, Janet M

    2003-01-01

    The first crucial step in any structural genomics project is the selection and prioritization of target proteins for structure determination. There may be a number of selection criteria to be satisfied, including that the proteins have novel folds, that they be representatives of large families for which no structure is known, and so on. The better the selection at this stage, the greater is the value of the structures obtained at the end of the experimental process. This value can be further enhanced once the protein structures have been solved if the functions of the given proteins can also be determined. Here we describe the methods used at either end of the experimental process: firstly, sensitive sequence comparison techniques for selecting a high-quality list of target proteins, and secondly the various computational methods that can be applied to the eventual 3D structures to determine the most likely biochemical function of the proteins in question. PMID:12880206

  5. Global and local attentional influences on target selection for action.

    PubMed

    McCarthy, J Daniel; Song, Joo-Hyun

    2015-09-01

    In daily life, humans interact with multiple objects in complex environments (e.g., fetching a glass from the cupboard, selecting a book on a shelf, etc.). Target selection is biased toward recently attended features, such that reaches are faster and trajectory curvature is reduced when target features (i.e., color) are repeated-a phenomenon referred to as "priming of pop-out". However, it is unclear how selecting a single target for action is impacted when it is grouped as part of a greater whole. We examined this question using a visually guided reaching task requiring participants to search for uniquely colored target among distractors and reach toward its location. Importantly, targets were Pac-men that were oriented to be either consistent or inconsistent with a global Kanizsa triangle. We found that movement initiation was faster when an illusory figure was present independent of color repetition and this effect increased with successive figure presentations. Additionally, movement duration and reach curvature were reduced when colors were repeated irrespective of configuration, consistent with priming of pop-out. An interaction also emerged for curvature: color switches were less costly when the configuration changed as well. We interpret this as evidence of binding between target color and global structure. To summarize, repeated Kanizsa figure presentations induced an early global priming effect indicated by faster movement initiation, whereas local color feature priming reduced movement duration and curvature, reflecting later focal decision processes. These results demonstrate that global and local attentional mechanisms play distinct roles in target selection for action and impact behavior during different stages of the decision process. Meeting abstract presented at VSS 2015. PMID:26326843

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

    PubMed Central

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

    1997-01-01

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

  7. Perceptual task induces saccadic adaptation by target selection.

    PubMed

    Schütz, Alexander C; Souto, David

    2015-01-01

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

  8. Perceptual task induces saccadic adaptation by target selection

    PubMed Central

    Schütz, Alexander C.; Souto, David

    2015-01-01

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

  9. Lysosomal glycerophosphocholine phosphodiesterase in Tetrahymena.

    PubMed

    Florin-Christensen, J; Florin-Christensen, M

    1999-02-01

    The purification and characterization of a novel phosphodiesterase (PDE) is presented. The activity was detected in the extracellular medium of Tetrahymena thermophila cultures, by the release of p-nitrophenol from p-nitrophenylphosphocholine (PNPPC) with an acidic pH optimum. In cell homogenates, it is sedimentable, shows a latency similar to that of acid phosphatase and is co-secreted with this enzyme, indicating that it is a lysosomal hydrolase. PNPPC-PDE was purified to homogeneity from the extracellular medium, yielding a single band of 58 kD on SDS polyacrylamide gel electrophoresis. It catalyzed the release of glycerol from glycerophosphocholine (GPC) and GPC competitively inhibits degradation of PNPPC. We present further evidence indicating that the natural substrate for PNPPC-PDE is GPC. Thus, Tetrahymena becomes the first eukaryote in which a lysosomal GPC-PDE is observed. This finding provides a new pathway for the complete breakdown of phosphatidylcholine in a lysosomal medium. PMID:10205674

  10. Target Selection for the SDSS-III MARVELS Survey

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2011-01-01

    Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid ? peptide (A?) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including A?1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of A? pathology. Systemic PADK injections in APPSwInd and APPswe/PS1?E9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both A? immunostaining as well as A?x-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive A? deposition in 20-22-month APPswe/PS1?E9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic A?1–38 occurs as A?1–42 levels decrease in the mouse models, indicating that PADK treatment leads to A? truncation. Associated with A? clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces A?1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of A? pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders. PMID:21695208

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

    SciTech Connect

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

    2011-03-10

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

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

    SciTech Connect

    Bertozzi, C.R.

    1997-12-31

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

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

    SciTech Connect

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

    2010-04-20

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

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

    PubMed

    Cubo, Ruben; Astrom, Mattias; Medvedev, Alexander

    2014-08-01

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

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

    PubMed

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

    2011-02-28

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

  1. A negative selection heuristic to predict new transcriptional targets

    PubMed Central

    2013-01-01

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

  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. Oncotripsy: Targeting cancer cells selectively via resonant harmonic excitation

    E-print Network

    Heyden, Stefanie

    2015-01-01

    We investigate a method of selectively targeting cancer cells by means of ultrasound harmonic excitation at their resonance frequency, which we refer to as oncotripsy. The geometric model of the cells takes into account the cytoplasm, nucleus and nucleolus, as well as the plasma membrane and nuclear envelope. Material properties are varied within a pathophysiologically-relevant range. A first modal analysis reveals the existence of a spectral gap between the natural frequencies and, most importantly, resonant growth rates of healthy and cancerous cells. The results of the modal analysis are verified by simulating the fully-nonlinear transient response of healthy and cancerous cells at resonance. The fully nonlinear analysis confirms that cancerous cells can be selectively taken to lysis by the application of carefully tuned ultrasound harmonic excitation while simultaneously leaving healthy cells intact.

  4. Selectively Searching for Conjunctively-Defined Visual Targets Charles E. Wright, April M. Main

    E-print Network

    White, Douglas R.

    MBS 96-14 Selectively Searching for Conjunctively-Defined Visual Targets Charles E. Wright, April M undergraduates) can search selectively through visual displays for a target defined by the conjunction of two

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Solitro, Abigail R; MacKeigan, Jeffrey P

    2016-01-01

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

  7. Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp).

    PubMed

    Jansson, Patric J; Yamagishi, Tetsuo; Arvind, Akanksha; Seebacher, Nicole; Gutierrez, Elaine; Stacy, Alexandra; Maleki, Sanaz; Sharp, Danae; Sahni, Sumit; Richardson, Des R

    2015-04-10

    Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity. PMID:25720491

  8. Target Selection for the SDSS-III MARVELS Survey

    E-print Network

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

    2015-01-01

    We present the target selection process for the Multi-object APO Radial Velocity Exoplanets Large-area Survey (MARVELS), which is part of the Sloan Digital Sky Survey (SDSS) III. MARVELS is a medium-resolution ($R \\sim 11000$) multi-fiber spectrograph capable of obtaining radial velocities for 60 objects at a time in order to find brown dwarfs and giant planets. The survey was configured to target dwarf stars with effective temperatures approximately between $4500$ and $6250 \\, \\mbox{K}$. For the first 2 years MARVELS relied on low-resolution spectroscopic pre-observations to estimate the effective temperature and $\\log(g)$ for candidate stars and then selected suitable dwarf stars from this pool. Ultimately, the pre-observation spectra proved ineffective at filtering out giant stars; many giants were incorrectly classified as dwarfs, resulting in a giant contamination rate of $\\sim$30\\% for the first phase of the MARVELS survey. Thereafter, the survey instead applied a reduced proper motion cut to eliminate ...

  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. Landslide susceptibility mapping in three selected target zones in Afghanistan

    NASA Astrophysics Data System (ADS)

    Schwanghart, Wolfgang; Seegers, Joe; Zeilinger, Gerold

    2015-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  12. Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to

    E-print Network

    Xu, Haoxing

    20850 Edited by David E. Clapham, Howard Hughes Medical Institute, Boston Children's Hospital, Boston as TRPML1 or ML1), a Ca2+ channel in the lysosome that regulates many aspects of lysosomal trafficking

  13. Subtype-selective targeting of voltage-gated sodium channels

    PubMed Central

    England, Steve; de Groot, Marcel J

    2009-01-01

    Voltage-gated sodium channels are key to the initiation and propagation of action potentials in electrically excitable cells. Molecular characterization has shown there to be nine functional members of the family, with a high degree of sequence homology between the channels. This homology translates into similar biophysical and pharmacological properties. Confidence in some of the channels as drug targets has been boosted by the discovery of human mutations in the genes encoding a number of them, which give rise to clinical conditions commensurate with the changes predicted from the altered channel biophysics. As a result, they have received much attention for their therapeutic potential. Sodium channels represent well-precedented drug targets as antidysrhythmics, anticonvulsants and local anaesthetics provide good clinical efficacy, driven through pharmacology at these channels. However, electrophysiological characterization of clinically useful compounds in recombinant expression systems shows them to be weak, with poor selectivity between channel types. This has led to the search for subtype-selective modulators, which offer the promise of treatments with improved clinical efficacy and better toleration. Despite developments in high-throughput electrophysiology platforms, this has proven very challenging. Structural biology is beginning to offer us a greater understanding of the three-dimensional structure of voltage-gated ion channels, bringing with it the opportunity to do real structure-based drug design in the future. This discipline is still in its infancy, but developments with the expression and purification of prokaryotic sodium channels offer the promise of structure-based drug design in the not too distant future. PMID:19845672

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-02-01

    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. Recent advances in gene therapy for lysosomal storage disorders

    PubMed Central

    Rastall, David PW; Amalfitano, Andrea

    2015-01-01

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

  19. Promotion of PDT efficacy by low-dose lysosomal photodamage

    NASA Astrophysics Data System (ADS)

    Kessel, David

    2015-03-01

    There have been literature reports indicating that protocols involving two photosensitizing agents, in animal tumor models, can yield a synergistic result, i.e., more photokilling than can be obtained with either sensitizer alone at the same light dose. We have independently obtained similar results in a cell-culture system that permits a more detailed study of mechanisms involved. Using any of three agents that localize in lysosomes, we were able to show that low-dose lysosomal photodamage could substantially promote photokilling by benzoporphyrin derivative, an agent that primarily targets mitochondria. This effect was abolished by knockdowns of either of two genes normally associated with autophagy: ATG5 and ATG7. A mechanism that can account for these results is proposed.

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

    PubMed

    Blume-Peytavi, U; Vogt, A

    2011-10-01

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

  1. 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

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

    PubMed

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

    2015-01-01

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

  3. A TRP Channel Senses Lysosome Neutralization by Pathogens to Trigger Their Expulsion.

    PubMed

    Miao, Yuxuan; Li, Guojie; Zhang, Xiaoli; Xu, Haoxing; Abraham, Soman N

    2015-06-01

    Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens. PMID:26027738

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

    PubMed Central

    Oh, Doo-Byoung

    2015-01-01

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

  5. 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

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

    SciTech Connect

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

    2009-11-01

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

  7. 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 cell hypothesis suggests that, unlike most cancer cells within a tumor, cancer stem cells resist of the disease. Thus, drugs that selectively target cancer stem cells offer great promise for cancer treatment

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

  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)kg day(-1) and 0.47 (0.03)kg day(-1), respectively (P=0.41). Thus, performance in dairy calves can potentially be maintained with fewer anthelmintic treatments but farmers need to be vigilant of the challenge posed by lungworm. Any future approach into the use of TST in FGS calves must take into consideration the relative importance of lungworm as a pathogen. PMID:25770853

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

    PubMed

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

    2016-01-15

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

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

    PubMed Central

    Ingemann, Linda; Kirkegaard, Thomas

    2014-01-01

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

  12. Bimanual Interference Associated With the Selection of Target Locations Jorn Diedrichsen and Richard B. Ivry

    E-print Network

    Jacobs, Lucia

    the preparation of bimanual reaching movements. Target locations were specified by color, and the right-hand and left-hand targets could be either the same or a different color. Movements of different amplitudes during bimanual movements arise during target selection rather than during motor programming. Experiments

  13. UNCORRECTED 2 Target selection in visual search as revealed by movement trajectories

    E-print Network

    Nakayama, Ken

    UNCORRECTED PROOF 1 2 Target selection in visual search as revealed by movement trajectories 3 Joo movements in visual search, in which participants reached to an odd-col- 9 ored target presented with two between red and green, and the location of the target was varied. Therefore either color could

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

    PubMed Central

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

    2015-01-01

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

  15. Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl

    E-print Network

    Bogyo, Matthew

    Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease selective, cell-permeable reagents for monitoring legumain activity in complex proteomes. Ó 2006 Elsevier B cell lysates9 . However, this reagent lacks cell permeability and its overall selectivity toward

  16. Targets of Balancing Selection in the Human Genome Aida M. Andres,* 1

    E-print Network

    Nielsen, Rasmus

    , it is a key force affecting the evolution of a number of genes in humans. Introduction Balancing selectionTargets of Balancing Selection in the Human Genome Aida M. Andre´s,* 1 Melissa J. Hubisz,à Amit; and #Department of Statistics, University of California, Berkeley Balancing selection is potentially an important

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

    PubMed Central

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

    2011-01-01

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

  18. Regional Personalized Electrodes to Select Transcranial Current Stimulation Target

    PubMed Central

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

    2013-01-01

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

  19. The ELG target selection with the BOSS survey

    NASA Astrophysics Data System (ADS)

    Escoffier, S.; Comparat, J.; Ealet, A.; Kneib, J.-P.; Zoubian, J.; Lamareille, F.

    2012-12-01

    The Baryon Acoustic Oscillation (BAO) feature in the power spectrum of galaxies can be used as a standard ruler to probe the accelerated expansion of the Universe. In this paper, we study several galaxy selection schemes aiming at building an emission-line galaxy (ELG) sample in the redshift range 0.6 < z < 1.7, that would be suitable for future BAO studies using the Baryonic Oscillation Spectroscopic Survey (BOSS) spectrograph on the Sloan Digital Sky Survey (SDSS) telescope. We explore two different color selections using both the SDSS and the Canada-France-Hawaii Telescope Legacy Survey (CFHTLS) photometry in the u, g, r, i bands and evaluate their performance for selecting bright ELG. This study confirms the feasibility of massive ELG surveys using the BOSS spectrographs on the SDSS telescope for a BAO detection at redshift z˜1, in particular for the proposed eBOSS experiment.

  20. 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

  1. Target selection by natural and redesigned PUF proteins.

    PubMed

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

    2015-12-29

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

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

    NASA Technical Reports Server (NTRS)

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

    1976-01-01

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

  3. A classifier-based target cost for unit selection speech synthesis trained on perceptual data 

    E-print Network

    Strom, Volker; King, Simon

    2010-01-01

    Our goal is to automatically learn a PERCEPTUALLY-optimal target cost function for a unit selection speech synthesiser. The approach we take here is to train a classifier on human perceptual judgements of synthetic speech. ...

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-06-15

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

  6. 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

  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. A new lactoferrin- and iron-dependent lysosomal death pathway is induced by benzo[a]pyrene in hepatic epithelial cells

    SciTech Connect

    Gorria, Morgane; Tekpli, Xavier; Rissel, Mary |; Sergent, Odile; Huc, Laurence |; Landvik, Nina; Fardel, Olivier; Dimanche-Boitrel, Marie-Therese |; Holme, Jorn A.; Lagadic-Gossmann, Dominique |

    2008-04-15

    While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation and cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.

  9. 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

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

    PubMed

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

    2015-11-18

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

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

    PubMed

    Sadekar, S; Figueroa, I; Tabrizi, M

    2015-07-01

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

  12. Neuraminidase 1 is a Negative Regulator of Lysosomal Exocytosis

    PubMed Central

    Yogalingam, Gouri; Bonten, Erik J.; van de Vlekkert, Diantha; Hu, Huimin; Moshiach, Simon; Connell, Samuel A.; d’Azzo, Alessandra

    2009-01-01

    SUMMARY Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase Neu1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein Lamp-1. In macrophages from Neu1-deficient mice, a model of the disease sialidosis, and in patients’ fibroblasts, oversialylated Lamp-1 enhances lysosomal exocytosis. Silencing of Lamp-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In Neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases. PMID:18606142

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

    PubMed

    Hu, Ye; Bajorath, Jürgen

    2015-01-01

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

  14. Quantifying the Tendency of Therapeutic Target Proteins to Bind Promiscuous or Selective Compounds

    PubMed Central

    Hu, Ye; Bajorath, Jürgen

    2015-01-01

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

  15. Development of target protein-selective degradation inducer for protein knockdown.

    PubMed

    Itoh, Yukihiro; Ishikawa, Minoru; Kitaguchi, Risa; Sato, Shinichi; Naito, Mikihiko; Hashimoto, Yuichi

    2011-05-15

    Our previous technique for inducing selective degradation of target proteins with ester-type SNIPER (Specific and Nongenetic Inhibitor-of-apoptosis-proteins (IAPs)-dependent Protein ERaser) degrades both the target proteins and IAPs. Here, we designed a small-molecular amide-type SNIPER to overcome this issue. As proof of concept, we synthesized and biologically evaluated an amide-type SNIPER which induces selective degradation of cellular retinoic acid binding protein II (CRABP-II), but not IAPs. Such small-molecular, amide-type SNIPERs that induce target protein-selective degradation without affecting IAPs should be effective tools to study the biological roles of target proteins in living cells. PMID:21515062

  16. Lysosomal storage diseases--the horizon expands.

    PubMed

    Boustany, Rose-Mary Naaman

    2013-10-01

    Since the discovery of the lysosome in 1955, advances have been made in understanding the key roles and functions of this organelle. The concept of lysosomal storage diseases (LSDs)--disorders characterized by aberrant, excessive storage of cellular material in lysosomes--developed following the discovery of ?-glucosidase deficiency as the cause of Pompe disease in 1963. Great strides have since been made in understanding the pathobiology of LSDs and the neuronal ceroid lipofuscinoses (NCLs). The NCLs are neurodegenerative disorders that display symptoms of cognitive and motor decline, seizures, blindness, early death, and accumulation of lipofuscin in various cell types, and also show some similarities to 'classic' LSDs. Defective lysosomal storage can occur in many cell types, but the CNS and PNS are particularly vulnerable to LSDs and NCLs, being affected in two-thirds of these disorders. Most LSDs are inherited in an autosomal recessive manner, with the exception of X-linked Hunter disease, Fabry disease and Danon disease, and a variant type of adult NCL (Kuf disease). This Review provides a summary of known LSDs, and the pathways affected in these disorders. Existing therapies and barriers to development of novel and improved treatments for LSDs and NCLs are also discussed. PMID:23938739

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Close encounters of the lysosome/peroxisome kind

    PubMed Central

    Jin, Yui; Strunk, Bethany S.; Weisman, Lois S.

    2015-01-01

    Lysosomes provide a major source for cellular cholesterol; however, most of this cholesterol is trafficked to the plasma membrane via unknown mechanisms. In this issue of Cell, Chu et al. identify an unexpected role for peroxisomes in the transport of cholesterol from the lysosome to the plasma membrane via a lysosome-peroxisome membrane contact site. PMID:25860602

  19. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

    E-print Network

    Settembre, Carmine

    The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. ...

  20. Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein*

    PubMed Central

    Karaca, Ilker; Tamboli, Irfan Y.; Glebov, Konstantin; Richter, Josefine; Fell, Lisa H.; Grimm, Marcus O.; Haupenthal, Viola J.; Hartmann, Tobias; Gräler, Markus H.; van Echten-Deckert, Gerhild; Walter, Jochen

    2014-01-01

    Progressive accumulation of the amyloid ? protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid ? is generated during sequential cleavage of the amyloid precursor protein (APP) by ?- and ?-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of ?-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca2+ from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP. PMID:24808180

  1. Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases.

    PubMed

    Petit, Constance S; Roczniak-Ferguson, Agnes; Ferguson, Shawn M

    2013-09-30

    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

  2. Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain

    E-print Network

    Bogyo, Matthew

    labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have rights reserved. Legumain is a lysosomal cysteine protease that is conserved in diverse cell types including, plants, invertebrate parasites and mammals. It has a high propensity to cleave protein substrates

  3. Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells

    PubMed Central

    Zou, Jianlong; Hu, Bo; Arpag, Sezgi; Yan, Qing; Hamilton, Audra; Zeng, Yuan-Shan; Vanoye, Carlos G.

    2015-01-01

    Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5?-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4?/?) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4?/? cells. The fission defect was associated with a robust increase of intralysosomal Ca2+ in Fig4?/? cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca2+ efflux of lysosomes because the endogenous ligand of lysosomal Ca2+ channel TRPML1 is PI3,5P2 that is deficient in Fig4?/? cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca2+ level and rescued abnormal lysosomal storage in Fig4?/? culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca2+ efflux in Fig4?/? culture cells and Fig4?/? mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency. PMID:25926456

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

    NASA Astrophysics Data System (ADS)

    Campos, Daniel; Méndez, Vicenç; Bartumeus, Frederic

    2012-01-01

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

  5. 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

  6. 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.

  7. LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation

    PubMed Central

    Milkereit, Ruth; Persaud, Avinash; Vanoaica, Liviu; Guetg, Adriano; Verrey, Francois; Rotin, Daniela

    2015-01-01

    Mammalian target of rapamycin 1 (mTORC1), a master regulator of cellular growth, is activated downstream of growth factors, energy signalling and intracellular essential amino acids (EAAs) such as Leu. mTORC1 activation occurs at the lysosomal membrane, and involves V-ATPase stimulation by intra-lysosomal EAA (inside-out activation), leading to activation of the Ragulator, RagA/B-GTP and mTORC1 via Rheb-GTP. How Leu enters the lysosomes is unknown. Here we identified the lysosomal protein LAPTM4b as a binding partner for the Leu transporter, LAT1-4F2hc (SLC7A5-SLAC3A2). We show that LAPTM4b recruits LAT1-4F2hc to lysosomes, leading to uptake of Leu into lysosomes, and is required for mTORC1 activation via V-ATPase following EAA or Leu stimulation. These results demonstrate a functional Leu transporter at the lysosome, and help explain the inside-out lysosomal activation of mTORC1 by Leu/EAA. PMID:25998567

  8. The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments

    PubMed Central

    1990-01-01

    Cytolytic lymphocytes contain specialized lytic granules whose secretion during cell-mediated cytolysis results in target cell death. Using serial section EM of RNK-16, a natural killer cell line, we show that there are structurally distinct types of granules. Each type is composed of varying proportions of a dense core domain and a multivesicular cortical domain. The dense core domains contain secretory proteins thought to play a role in cytolysis, including cytolysin and chondroitin sulfate proteoglycan. In contrast, the multivesicular domains contain lysosomal proteins, including acid phosphatase, alpha-glucosidase, cathepsin D, and LGP-120. In addition to their protein content, the lytic granules have other properties in common with lysosomes. The multivesicular regions of the granules have an acidic pH, comparable to that of endosomes and lysosomes. The granules take up exogenous cationized ferritin with lysosome-like kinetics, and this uptake is blocked by weak bases and low temperature. The multivesicular domains of the granules are rich in the 270-kD mannose-6-phosphate receptor, a marker which is absent from mature lysosomes but present in earlier endocytic compartments. Thus, the natural killer granules represent an unusual dual-function organelle, where a regulated secretory compartment, the dense core, is contained within a pre-lysosomal compartment, the multivesicular domain. PMID:2277062

  9. Lysosomal Sequestration Determines Intracellular Imatinib Levels.

    PubMed

    Burger, Herman; den Dekker, Alexander T; Segeletz, Sandra; Boersma, Antonius W M; de Bruijn, Peter; Debiec-Rychter, Maria; Taguchi, Takahiro; Sleijfer, Stefan; Sparreboom, Alex; Mathijssen, Ron H J; Wiemer, Erik A C

    2015-09-01

    The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (organic cation transporter 1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here, we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses, and apoptosis assays were performed to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562 and SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time-, dose-, temperature-, and energy-dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH4Cl, and the vacuolar ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Costaining experiments with LysoTracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib-mediated cell cycle arrest but significantly increased apoptosis in imatinib-sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression. PMID:26108972

  10. 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

  11. 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

  12. The SDSS-III Baryon Oscillation Spectroscopic Survey: Quasar Target Selection for Data Release Nine

    NASA Astrophysics Data System (ADS)

    Ross, Nicholas P.; Myers, Adam D.; Sheldon, Erin S.; Yèche, Christophe; Strauss, Michael A.; Bovy, Jo; Kirkpatrick, Jessica A.; Richards, Gordon T.; Aubourg, Éric; Blanton, Michael R.; Brandt, W. N.; Carithers, William C.; Croft, Rupert A. C.; da Silva, Robert; Dawson, Kyle; Eisenstein, Daniel J.; Hennawi, Joseph F.; Ho, Shirley; Hogg, David W.; Lee, Khee-Gan; Lundgren, Britt; McMahon, Richard G.; Miralda-Escudé, Jordi; Palanque-Delabrouille, Nathalie; Pâris, Isabelle; Petitjean, Patrick; Pieri, Matthew M.; Rich, James; Roe, Natalie A.; Schiminovich, David; Schlegel, David J.; Schneider, Donald P.; Slosar, Anže; Suzuki, Nao; Tinker, Jeremy L.; Weinberg, David H.; Weyant, Anya; White, Martin; Wood-Vasey, W. Michael

    2012-03-01

    The SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS), a five-year spectroscopic survey of 10,000 deg2, achieved first light in late 2009. One of the key goals of BOSS is to measure the signature of baryon acoustic oscillations (BAOs) in the distribution of Ly? absorption from the spectra of a sample of ~150,000 z > 2.2 quasars. Along with measuring the angular diameter distance at z ? 2.5, BOSS will provide the first direct measurement of the expansion rate of the universe at z > 2. One of the biggest challenges in achieving this goal is an efficient target selection algorithm for quasars in the redshift range 2.2 < z < 3.5, where their colors tend to overlap those of the far more numerous stars. During the first year of the BOSS survey, quasar target selection (QTS) methods were developed and tested to meet the requirement of delivering at least 15 quasars deg-2 in this redshift range, with a goal of 20 out of 40 targets deg-2 allocated to the quasar survey. To achieve these surface densities, the magnitude limit of the quasar targets was set at g <= 22.0 or r <= 21.85. While detection of the BAO signature in the distribution of Ly? absorption in quasar spectra does not require a uniform target selection algorithm, many other astrophysical studies do. We have therefore defined a uniformly selected subsample of 20 targets deg-2, for which the selection efficiency is just over 50% (~10 z > 2.20 quasars deg-2). This "CORE" subsample will be fixed for Years Two through Five of the survey. For the remaining 20 targets deg-2, we will continue to develop improved selection techniques, including the use of additional data sets beyond the Sloan Digital Sky Survey (SDSS) imaging data. In this paper, we describe the evolution and implementation of the BOSS QTS algorithms during the first two years of BOSS operations (through 2011 July), in support of the science investigations based on these data, and we analyze the spectra obtained during the first year. During this year, 11,263 new z > 2.20 quasars were spectroscopically confirmed by BOSS, roughly double the number of previously known quasars with z > 2.20. Our current algorithms select an average of 15 z > 2.20 quasars deg-2 from 40 targets deg-2 using single-epoch SDSS imaging. Multi-epoch optical data and data at other wavelengths can further improve the efficiency and completeness of BOSS QTS.

  13. Aptamer-CaCO3 nanostructures: A facile, pH-responsive, specific platform for targeted anticancer theranostics

    PubMed Central

    Zhou, Cuisong; Chen, Tao; Wu, Cuichen; Zhu, Guizhi; Qiu, Liping; Cui, Cheng; Hou, Weijia; Tan, Weihong

    2014-01-01

    Application of cancer theranostics depends on the development of multifunctional nanostructure platforms for accurate cell targeting and controlled drug release, imaging and therapy. In this work, a comprehensive, easily fabricated anticancer theranostic platform with high drug-loading capacity, termed aptamer-functionalized calcium carbonate (CaCO3) nanostructure (apt-CCN), is reported. Flow cytometry and confocal fluorescence microscopy studies demonstrated that apt-CCNs can specifically bind to target cancer cells, but not to control cells, and that they possess highly efficient internalization to target cancer cells. This smart nanostructure selectively reaches the lysosomes through receptor-mediated endocytosis and is responsive to the relatively low lysosome pH (4.5–5.5), facilitating the release of doxorubicin. The apt-CCN platform offers targeted and efficient drug transport, as well as target-specific delivery of imaging agents for cancer diagnosis and therapy. PMID:25377905

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

    SciTech Connect

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

    2013-10-01

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

  15. 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 describes and evaluates privacy-friendly methods for extracting quasi-social networks from browser behavior-generated content, privacy This work was conducted while the authors were at Media6 . Foster Provost thanks NEC

  16. Improving Selection of Off-Screen Targets with Hopping Pourang Irani

    E-print Network

    +context and overview+detail views, such as fisheye or radar views, have also been considered as ways to facilitate and cognitive costs (e.g., [10]). For the task of selecting off-screen targets, no current method is able

  17. MADS-box genes of maize: frequent targets of selection during domestication

    E-print Network

    Doebley, John

    MADS-box genes of maize: frequent targets of selection during domestication QIONG ZHAO1 MADS-box genes encode transcription factors that are key regulators of plant inflorescence and flower development. We examined DNA sequence variation in 32 maize MADS-box genes and 32 randomly chosen maize loci

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    E-print Network

    Janzen, Fredric

    Effective heritability of targets of sex-ratio selection under environmental sex determination S. E, 2008; Robinson et al., 2009). In many organisms, sex of the offspring is determined by environmental-by-environment interaction; nest-site choice; phenotypic plasticity; sex ratio; temperature-dependent sex determination

  1. 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…

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  5. Reduction of Nanoparticle Avidity Enhances the Selectivity of Vascular Targeting and PET Detection of Pulmonary Inflammation

    PubMed Central

    Zern, Blaine J.; Chacko, Ann-Marie; Liu, Jin; Greineder, Colin F.; Blankemeyer, Eric R.; Radhakrishnan, Ravi; Muzykantov, Vladimir

    2013-01-01

    Targeting nanoparticles (NPs) loaded with drugs and probes to precise locations in the body may improve the treatment and detection of many diseases. Generally, to achieve targeting, affinity ligands are introduced on the surface of NPs that can bind to molecules present on the cell of interest. Optimization of ligand density is a critical parameter in controlling NP binding to target cells and a higher ligand density is not always the most effective. In this study, we investigated how NP avidity affects targeting to the pulmonary vasculature, using NPs targeted to ICAM-1. This cell adhesion molecule is expressed by quiescent endothelium at modest levels and is upregulated in a variety of pathological settings. NP avidity was controlled by ligand density, with the expected result that higher avidity NPs demonstrated greater pulmonary uptake than lower avidity NPs in both naïve and pathological mice. However, in comparison with high avidity NPs, low avidity NPs exhibited several-fold higher selectivity of targeting to pathological endothelium. This finding was translated into a PET imaging platform that was more effective in detecting pulmonary vascular inflammation using low avidity NPs. Furthermore, computational modeling revealed that elevated expression of ICAM-1 on the endothelium is critical for multivalent anchoring of NPs with low avidity, while high avidity NPs anchor effectively to both quiescent and activated endothelium. These results provide a paradigm that can be used to optimize NP targeting by manipulating ligand density, and may find biomedical utility for increasing detection of pathological vasculature. PMID:23383962

  6. Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat

    PubMed Central

    Gagnon, Keith T.; Pendergraff, Hannah M.; Deleavey, Glen F.; Swayze, Eric E.; Potier, Pierre; Randolph, John; Roesch, Eric B.; Chattopadhyaya, Jyoti; Damha, Masad J.; Bennett, C. Frank; Montaillier, Christophe; Lemaitre, Marc; Corey, David R.

    2010-01-01

    Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD. PMID:21028906

  7. Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents

    PubMed Central

    Modica-Napolitano, Josephine S.; Weissig, Volkmar

    2015-01-01

    Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including those that induce mitochondria permeability transition and apoptosis, metabolic inhibitors, and ROS regulators. To date, however, none has exhibited the standards for high selectivity and efficacy and low toxicity necessary to progress beyond phase III clinical trials and be used as a viable, single modality treatment option for human cancers. This review explores alternative treatment strategies that have been shown to enhance the efficacy and selectivity of mitochondria-targeted anticancer agents in vitro and in vivo, and may yet fulfill the clinical promise of exploiting the mitochondrion as a target for cancer chemotherapy. PMID:26230693

  8. Diverse selective regimes shape genetic diversity at ADAR genes and at their coding targets.

    PubMed

    Forni, Diego; Mozzi, Alessandra; Pontremoli, Chiara; Vertemara, Jacopo; Pozzoli, Uberto; Biasin, Mara; Bresolin, Nereo; Clerici, Mario; Cagliani, Rachele; Sironi, Manuela

    2015-01-01

    A-to-I RNA editing operated by ADAR enzymes is extremely common in mammals. Several editing events in coding regions have pivotal physiological roles and affect protein sequence (recoding events) or function. We analyzed the evolutionary history of the 3 ADAR family genes and of their coding targets. Evolutionary analysis indicated that ADAR evolved adaptively in primates, with the strongest selection in the unique N-terminal domain of the interferon-inducible isoform. Positively selected residues in the human lineage were also detected in the ADAR deaminase domain and in the RNA binding domains of ADARB1 and ADARB2. During the recent history of human populations distinct variants in the 3 genes increased in frequency as a result of local selective pressures. Most selected variants are located within regulatory regions and some are in linkage disequilibrium with eQTLs in monocytes. Finally, analysis of conservation scores of coding editing sites indicated that editing events are counter-selected within regions that are poorly tolerant to change. Nevertheless, a minority of recoding events occurs at highly conserved positions and possibly represents the functional fraction. These events are enriched in pathways related to HIV-1 infection and to epidermis/hair development. Thus, both ADAR genes and their targets evolved under variable selective regimes, including purifying and positive selection. Pressures related to immune response likely represented major drivers of evolution for ADAR genes. As for their coding targets, we suggest that most editing events are slightly deleterious, although a minority may be beneficial and contribute to antiviral response and skin homeostasis. PMID:25826567

  9. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

    PubMed Central

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

  10. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells

    PubMed Central

    He, Huan; Li, Dong-Wei; Yang, Li-Yun; Fu, Li; Zhu, Xun-Jin; Wong, Wai-Kwok; Jiang, Feng-Lei; Liu, Yi

    2015-01-01

    Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. PMID:26337336

  11. Processing and activation of latent heparanase occurs in lysosomes.

    PubMed

    Zetser, Anna; Levy-Adam, Flonia; Kaplan, Victoria; Gingis-Velitski, Svetlana; Bashenko, Yulia; Schubert, Shay; Flugelman, Moshe Y; Vlodavsky, Israel; Ilan, Neta

    2004-05-01

    Heparanase is a heparan sulfate degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Heparanase is synthesized as a 65 kDa non-active precursor that subsequently undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The protease responsible for heparanase processing is currently unknown, as is the sub-cellular processing site. In this study, we characterize an antibody (733) that preferentially recognizes the active 50 kDa heparanase form as compared to the non-active 65 kDa heparanase precursor. We have utilized this and other anti-heparanase antibodies to study the cellular localization of the latent 65 kDa and active 50 kDa heparanase forms during uptake and processing of exogenously added heparanase. Interestingly, not only the processed 50 kDa, but also the 65 kDa heparanase precursor was localized to perinuclear vesicles, suggesting that heparanase processing occurs in lysosomes. Indeed, heparanase processing was completely inhibited by chloroquine and bafilomycin A1, inhibitors of lysosome proteases. Similarly, processing of membrane-targeted heparanase was also chloroquine-sensitive, further ruling out the plasma membrane as the heparanase processing site. Finally, we provide evidence that antibody 733 partially neutralizes the enzymatic activity of heparanase, suggesting that the N-terminal region of the molecule is involved in assuming an active conformation. Monoclonal antibodies directed to this region are likely to provide specific heparanase inhibitors and hence assist in resolving heparanase functions under normal and pathological conditions. PMID:15126626

  12. 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.

  13. 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

  14. The lysosomal enzyme receptor protein (LERP) is not essential, but is implicated in lysosomal function in Drosophila melanogaster

    PubMed Central

    Hasanagic, Medina; van Meel, Eline; Luan, Shan; Aurora, Rajeev; Kornfeld, Stuart; Eissenberg, Joel C.

    2015-01-01

    ABSTRACT The lysosomal enzyme receptor protein (LERP) of Drosophila melanogaster is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P) receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. Here, we analyze the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. We generated a novel Lerp null mutation, LerpF6, which abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30–40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. Our findings implicate Lerp in lysosome function and autophagy. PMID:26405051

  15. Impact of high-risk conjunctions on Active Debris Removal target selection

    NASA Astrophysics Data System (ADS)

    Lidtke, Aleksander A.; Lewis, Hugh G.; Armellin, Roberto

    2015-10-01

    Space debris simulations show that if current space launches continue unchanged, spacecraft operations might become difficult in the congested space environment. It has been suggested that Active Debris Removal (ADR) might be necessary in order to prevent such a situation. Selection of objects to be targeted by ADR is considered important because removal of non-relevant objects will unnecessarily increase the cost of ADR. One of the factors to be used in this ADR target selection is the collision probability accumulated by every object. This paper shows the impact of high-probability conjunctions on the collision probability accumulated by individual objects as well as the probability of any collision occurring in orbit. Such conjunctions cannot be predicted far in advance and, consequently, not all the objects that will be involved in such dangerous conjunctions can be removed through ADR. Therefore, a debris remediation method that would address such events at short notice, and thus help prevent likely collisions, is suggested.

  16. 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

  17. 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

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

    PubMed

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

    2010-05-01

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

  19. 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

  20. Functionalized paper SERS (P-SERS) substrates for selective targeting of analytes in complex samples

    NASA Astrophysics Data System (ADS)

    Yu, Wei W.; Hoppmann, Eric P.

    2015-05-01

    Surface enhanced Raman spectroscopy (SERS) requires the analyte molecule to be close to the plasmonic surface in order to generate SERS enhancement. This limitation restricts the practical application of SERS to molecules that possess functional groups that interact strongly with gold or silver surfaces. Moreover, the identification of target analytes in a complex sample matrix is made even more difficult when interferents compete with the target for binding to the plasmonic surface, resulting in overlapping spectral signatures. In this work, we report a strategy to functionalize inkjet printed P-SERS substrates by strategically placing supramolecular structures (such as nucleic acid aptamers) onto the gold nanoparticles. This promotes the selective interaction of target molecules with the plasmonic surface, leading to improved sensor performance.

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

    SciTech Connect

    Vinoth Kumar, L.; Manikanta, E.; Leela, Ch.; Prem Kiran, P.

    2014-08-11

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

  2. Selective targeting of indel-inferred differences in spatial structures of highly homologous proteins.

    PubMed

    Cherkasov, Artem; Nandan, Devki; Reiner, Neil E

    2005-03-01

    Recent findings have shown that the protein elongation factor-1alpha (EF-1alpha) from the eukaryotic pathogen Leishmania donovani possesses virulence properties. This was unexpected, since it has greater than 80% sequence identity with its human homologue. Given that EF-1alpha is essential for cell survival, in principle, it can be considered an attractive drug target. However, the challenge is to be able to selectively target the protein so as not to affect function of the human homologue. While a limited number of discrete differences were scattered throughout the sequence, most of the difference between these 2 homologues could be attributed to a 12-amino acid insert present in human EF-1alpha and absent from the leishmania sequence. In the present study, we modeled the spatial differences in structures of human and L. donovani EF-1alpha's inferred by this insertion-deletion (or "indel"). The protein models were used to develop antibodies directed specifically toward the deletion region of the pathogen protein. The strategy described allowed successful selective targeting of this putative leishmania virulence factor while avoiding recognition of the highly similar human EF-1alpha homologue. These findings may establish a new strategy for the development of antagonists directed against certain pathogenic targets having close human homologues. PMID:15657927

  3. Synthetic, Non-saccharide, Glycosaminoglycan Mimetics Selectively Target Colon Cancer Stem Cells

    PubMed Central

    2015-01-01

    Selective targeting of cancer stem-like cells (CSCs) is a paradigm-shifting approach. We hypothesized that CSCs can be targeted by interfering with functions of sulfated glycosaminoglycans, which play key roles in cancer cell growth, invasion and metastasis. We developed a tandem, dual screen strategy involving (1) assessing inhibition of monolayer versus spheroid growth and (2) assessing inhibition of primary versus secondary spheroid growth to identify G2.2, a unique sulfated nonsaccharide GAG mimetic (NSGM) from a focused library of 53 molecules, as a selective inhibitor of colon CSCs. The NSGM down-regulated several CSC markers through regulation of gene transcription, while closely related, inactive NSGMs G1.4 and G4.1 demonstrated no such changes. G2.2’s effects on CSCs were mediated, in part, through induction of apoptosis and inhibition of self-renewal factors. Overall, this work presents the proof-of-principle that CSCs can be selectively targeted through novel NSGMs, which are likely to advance fundamental understanding on CSCs while also aiding development of novel therapeutic agents. PMID:24968014

  4. The Niemann-Pick C1 Protein Functions in Regulating Lysosome Amine Content

    E-print Network

    Kaufmann, Allyn

    2008-01-01

    Mutations in the lysosome membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport from the lysosome, resulting in the hyper-accumulation of multiple lysosomal ...

  5. THE SDSS-III BARYON OSCILLATION SPECTROSCOPIC SURVEY: QUASAR TARGET SELECTION FOR DATA RELEASE NINE

    SciTech Connect

    Ross, Nicholas P.; Kirkpatrick, Jessica A.; Carithers, William C.; Ho, Shirley; Myers, Adam D.; Sheldon, Erin S.; Yeche, Christophe; Aubourg, Eric; Strauss, Michael A.; Lee, Khee-Gan; Bovy, Jo; Blanton, Michael R.; Hogg, David W.; Richards, Gordon T.; Brandt, W. N.; Croft, Rupert A. C.; Da Silva, Robert; Dawson, Kyle; Eisenstein, Daniel J.; Hennawi, Joseph F.; and others

    2012-03-01

    The SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS), a five-year spectroscopic survey of 10,000 deg{sup 2}, achieved first light in late 2009. One of the key goals of BOSS is to measure the signature of baryon acoustic oscillations (BAOs) in the distribution of Ly{alpha} absorption from the spectra of a sample of {approx}150,000 z > 2.2 quasars. Along with measuring the angular diameter distance at z Almost-Equal-To 2.5, BOSS will provide the first direct measurement of the expansion rate of the universe at z > 2. One of the biggest challenges in achieving this goal is an efficient target selection algorithm for quasars in the redshift range 2.2 < z < 3.5, where their colors tend to overlap those of the far more numerous stars. During the first year of the BOSS survey, quasar target selection (QTS) methods were developed and tested to meet the requirement of delivering at least 15 quasars deg{sup -2} in this redshift range, with a goal of 20 out of 40 targets deg{sup -2} allocated to the quasar survey. To achieve these surface densities, the magnitude limit of the quasar targets was set at g {<=} 22.0 or r {<=} 21.85. While detection of the BAO signature in the distribution of Ly{alpha} absorption in quasar spectra does not require a uniform target selection algorithm, many other astrophysical studies do. We have therefore defined a uniformly selected subsample of 20 targets deg{sup -2}, for which the selection efficiency is just over 50% ({approx}10 z > 2.20 quasars deg{sup -2}). This 'CORE' subsample will be fixed for Years Two through Five of the survey. For the remaining 20 targets deg{sup -2}, we will continue to develop improved selection techniques, including the use of additional data sets beyond the Sloan Digital Sky Survey (SDSS) imaging data. In this paper, we describe the evolution and implementation of the BOSS QTS algorithms during the first two years of BOSS operations (through 2011 July), in support of the science investigations based on these data, and we analyze the spectra obtained during the first year. During this year, 11,263 new z > 2.20 quasars were spectroscopically confirmed by BOSS, roughly double the number of previously known quasars with z > 2.20. Our current algorithms select an average of 15 z > 2.20 quasars deg{sup -2} from 40 targets deg{sup -2} using single-epoch SDSS imaging. Multi-epoch optical data and data at other wavelengths can further improve the efficiency and completeness of BOSS QTS.

  6. 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.

  7. A Two-Step Target Binding and Selectivity Support Vector Machines Approach for Virtual Screening of Dopamine Receptor Subtype-Selective Ligands

    PubMed Central

    Zhang, Jingxian; Han, Bucong; Wei, Xiaona; Tan, Chunyan; Chen, Yuzong; Jiang, Yuyang

    2012-01-01

    Target selective drugs, such as dopamine receptor (DR) subtype selective ligands, are developed for enhanced therapeutics and reduced side effects. In silico methods have been explored for searching DR selective ligands, but encountered difficulties associated with high subtype similarity and ligand structural diversity. Machine learning methods have shown promising potential in searching target selective compounds. Their target selective capability can be further enhanced. In this work, we introduced a new two-step support vector machines target-binding and selectivity screening method for searching DR subtype-selective ligands, which was tested together with three previously-used machine learning methods for searching D1, D2, D3 and D4 selective ligands. It correctly identified 50.6%–88.0% of the 21–408 subtype selective and 71.7%–81.0% of the 39–147 multi-subtype ligands. Its subtype selective ligand identification rates are significantly better than, and its multi-subtype ligand identification rates are comparable to the best rates of the previously used methods. Our method produced low false-hit rates in screening 13.56 M PubChem, 168,016 MDDR and 657,736 ChEMBLdb compounds. Molecular features important for subtype selectivity were extracted by using the recursive feature elimination feature selection method. These features are consistent with literature-reported features. Our method showed similar performance in searching estrogen receptor subtype selective ligands. Our study demonstrated the usefulness of the two-step target binding and selectivity screening method in searching subtype selective ligands from large compound libraries. PMID:22720033

  8. A two-step target binding and selectivity support vector machines approach for virtual screening of dopamine receptor subtype-selective ligands.

    PubMed

    Zhang, Jingxian; Han, Bucong; Wei, Xiaona; Tan, Chunyan; Chen, Yuzong; Jiang, Yuyang

    2012-01-01

    Target selective drugs, such as dopamine receptor (DR) subtype selective ligands, are developed for enhanced therapeutics and reduced side effects. In silico methods have been explored for searching DR selective ligands, but encountered difficulties associated with high subtype similarity and ligand structural diversity. Machine learning methods have shown promising potential in searching target selective compounds. Their target selective capability can be further enhanced. In this work, we introduced a new two-step support vector machines target-binding and selectivity screening method for searching DR subtype-selective ligands, which was tested together with three previously-used machine learning methods for searching D1, D2, D3 and D4 selective ligands. It correctly identified 50.6%-88.0% of the 21-408 subtype selective and 71.7%-81.0% of the 39-147 multi-subtype ligands. Its subtype selective ligand identification rates are significantly better than, and its multi-subtype ligand identification rates are comparable to the best rates of the previously used methods. Our method produced low false-hit rates in screening 13.56 M PubChem, 168,016 MDDR and 657,736 ChEMBLdb compounds. Molecular features important for subtype selectivity were extracted by using the recursive feature elimination feature selection method. These features are consistent with literature-reported features. Our method showed similar performance in searching estrogen receptor subtype selective ligands. Our study demonstrated the usefulness of the two-step target binding and selectivity screening method in searching subtype selective ligands from large compound libraries. PMID:22720033

  9. 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…

  10. SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm.

    PubMed

    Hamblett, Kevin J; Jacob, Allison P; Gurgel, Jesse L; Tometsko, Mark E; Rock, Brooke M; Patel, Sonal K; Milburn, Robert R; Siu, Sophia; Ragan, Seamus P; Rock, Dan A; Borths, Christopher J; O'Neill, Jason W; Chang, Wesley S; Weidner, Margaret F; Bio, Matthew M; Quon, Kim C; Fanslow, William C

    2015-12-15

    Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens. Cancer Res; 75(24); 5329-40. ©2015 AACR. PMID:26631267

  11. 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

  12. 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.

  13. Selective targeting of the retinal pigment epithelium using an acousto-optic laser scanner.

    PubMed

    Alt, Clemens; Framme, Carsten; Schnell, Susanne; Lee, Ho; Brinkmann, Ralf; Lin, Charles P

    2005-01-01

    Selective targeting of the retinal pigment epithelium (RPE) is a new strategy for treating certain retinal disorders while preserving adjacent photoreceptors. The treatment currently relies on a complex laser system to produce the required microsecond pulse structure. In our new approach, we scan the focus of a continuous-wave (cw) laser beam with acousto-optic deflectors to produce microsecond-long exposures at each RPE cell. Experiments were performed in vitro with a bench-top scanner on samples of young bovine RPE and in vivo on Dutch belted rabbits with a slit-lamp adapted scanner. Effective dose 50% (ED50) for RPE damage was determined in vitro by fluorescence cell viability assay and in vivo by fluorescein angiography. Damage to individual RPE cells was achieved with laser power on the order of 100 mW. Using separated scan lines, we demonstrate selectivity in the form of alternating lines of dead and surviving cells that resemble the scan pattern. Selectivity is also shown by the absence of retinal thermal coagulation in vivo. Selective RPE damage is feasible by rapidly scanning a cw laser beam. The scanning device is an attractive alternative to conventional laser coagulation and pulsed laser targeting of the RPE. PMID:16409079

  14. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3K?-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  15. Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway.

    PubMed

    Fu, Ying-Zi; Yan, Yuan-Yuan; He, Miao; Xiao, Qing-Huan; Yao, Wei-Fan; Zhao, Lin; Wu, Hui-Zhe; Yu, Zhao-Jin; Zhou, Ming-Yi; Lv, Mu-Tian; Zhang, Shan-Shan; Chen, Jian-Jun; Wei, Min-Jie

    2016-02-01

    Breast cancer stem cells (BCSCs) are believed to be responsible for tumor chemoresistance, recurrence, and metastasis formation. Salinomycin (SAL), a carboxylic polyether ionophore, has been reported to act as a selective breast CSC inhibitor. However, the molecular mechanisms underlying SAL-induced cytotoxicity on BCSCs remain unclear. The Hedgehog (Hh) signaling pathway plays an important role in CSC maintenance and carcinogenesis. Here, we investigated whether SAL induces cytotoxicity on BCSCs through targeting Hh pathway. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain breast CSC-enriched MCF-7 mammospheres (MCF-7 MS). MCF-7 MS cells possessed typical BCSC properties, such as CD44+CD24-/low phenotype, high expression of OCT4 (a stem cell marker), increased colony-forming ability, strong migration and invasion capabilities, differentiation potential, and strong tumorigenicity in xenografted mice. SAL exhibited selective cytotoxicity to MCF-7 MS cells relative to MCF-7 cells. The Hh pathway was highly activated in BCSC-enriched MCF-7 MS cells and SAL inhibited Hh signaling activation by downregulating the expression of critical components of the Hh pathway such as PTCH, SMO, Gli1, and Gli2, and subsequently repressing the expression of their essential downstream targets including C-myc, Bcl-2, and Snail (but not cyclin D1). Conversely, Shh-induced Hh signaling activation could largely reverse SAL-mediated inhibitory effects. These findings suggest that SAL-induced selective cytotoxicity against MCF-7 MS cells is associated with the inhibition of Hh signaling activation and the expression of downstream targets and the Hh pathway is an important player and a possible drug target in the pathogenesis of BCSCs. PMID:26718029

  16. 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

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

    PubMed Central

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

    2009-01-01

    Background Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I 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 on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. Conclusions In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level. PMID:19158951

  18. Selective autophagy against membranous compartments

    PubMed Central

    Pimentel-Muiños, Felipe X; Boada-Romero, Emilio

    2014-01-01

    Selective autophagic degradation of cellular components underlies many of the important physiological and pathological implications that autophagy has for mammalian cells. Cytoplasmic vesicles, just like other intracellular items, can be subjected to conventional autophagic events where double-membrane autophagosomes specifically isolate and deliver them for lysosomal destruction. However, intracellular membranes appear to constitute common platforms for unconventional versions of the autophagic pathway, a notion that has become apparent during the past few years. For instance, in many cases of autophagy directed against bacterial phagosomes, subversion of the process results in multimembrane vacuoles that promote bacterial replication instead of the usual degradative outcome. In a different atypical modality, single-membrane vesicles can be labeled with LC3 to direct their contents for lysosomal degradation. In fact, single-membrane compartments of various kinds often provide an assembly site for the autophagic machinery to perform unanticipated nondegradative activities that range from localized secretion of lysosomal contents to melanosome function. Interestingly, many of these unconventional processes seem to be initiated through engagement of relevant nodes of the autophagic signaling network that, once activated, promote LC3 decoration of the targeted membrane, and some cases of inducer/receptor proteins that specifically engage those important signaling hubs have recently been described. Here we review the available examples of all autophagic variants involving membranous compartments, with a main focus on the more recently discovered unconventional phenomena where the usual degradation purpose of autophagy or its canonical mechanistic features are not completely conserved. PMID:24419294

  19. Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

    PubMed

    Abdul-Hay, Samer O; Bannister, Thomas D; Wang, Hui; Cameron, Michael D; Caulfield, Thomas R; Masson, Amandine; Bertrand, Juliette; Howard, Erin A; McGuire, Michael P; Crisafulli, Umberto; Rosenberry, Terrone R; Topper, Caitlyn L; Thompson, Caroline R; Schürer, Stephan C; Madoux, Franck; Hodder, Peter; Leissring, Malcolm A

    2015-12-18

    Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 ?M, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets. PMID:26398879

  20. 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

  1. The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

    PubMed Central

    Bird, C H; Christensen, M E; Mangan, M S J; Prakash, M D; Sedelies, K A; Smyth, M J; Harper, I; Waterhouse, N J; Bird, P I

    2014-01-01

    Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations. PMID:24488096

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

    SciTech Connect

    Carrer, H.; Maliga, P.

    1995-08-01

    To determine whether targeted DNA insertion into the tobacco plastid genome can be obtained without physical linkage to a selectable marker gene, we carried out biolistic transformation of chloroplasts in tobacco leaf segments with a 1:1 mix of two independently targeted antibiotic resistance genes. Plastid transformants were selected by spectinomycin resistance due to expression of an integrated aadA gene. Integration of the unselected kanamycin resistance (kan) gene into the same plastid genome was established by Southern probing in {approx}20% of the spectinomycin-selected clones. Efficient cotransformation will facilitate targeted plastid genome modification without physical linkage to a marker gene. 26 refs., 5 figs., 1 tab.

  3. Selective Vitamin D Receptor Activation as Anti-Inflammatory Target in Chronic Kidney Disease

    PubMed Central

    Donate-Correa, J.; Domínguez-Pimentel, V.; Méndez-Pérez, M. L.; Muros-de-Fuentes, M.; Mora-Fernández, C.; Martín-Núñez, E.; Cazaña-Pérez, V.; Navarro-González, J. F.

    2014-01-01

    Paricalcitol, a selective vitamin D receptor (VDR) activator used for treatment of secondary hyperparathyroidism in chronic kidney disease (CKD), has been associated with survival advantages, suggesting that this drug, beyond its ability to suppress parathyroid hormone, may have additional beneficial actions. In this prospective, nonrandomised, open-label, proof-of-concept study, we evaluated the hypothesis that selective vitamin D receptor activation with paricalcitol is an effective target to modulate inflammation in CKD patients. Eight patients with an estimated glomerular filtration rate between 15 and 44?mL/min/1.73?m2 and an intact parathyroid hormone (PTH) level higher than 110?pg/mL received oral paricalcitol (1??g/48 hours) as therapy for secondary hyperparathyroidism. Nine patients matched by age, sex, and stage of CKD, but a PTH level <110?pg/mL, were enrolled as a control group. Our results show that five months of paricalcitol administration were associated with a reduction in serum concentrations of hs-CRP (13.9%, P < 0.01), TNF-? (11.9%, P = 0.01), and IL-6 (7%, P < 0.05), with a nonsignificant increase of IL-10 by 16%. In addition, mRNA expression levels of the TNF? and IL-6 genes in peripheral blood mononuclear cells decreased significantly by 30.8% (P = 0.01) and 35.4% (P = 0.01), respectively. In conclusion, selective VDR activation is an effective target to modulate inflammation in CKD. PMID:24511210

  4. Selective vitamin D receptor activation as anti-inflammatory target in chronic kidney disease.

    PubMed

    Donate-Correa, J; Domínguez-Pimentel, V; Méndez-Pérez, M L; Muros-de-Fuentes, M; Mora-Fernández, C; Martín-Núñez, E; Cazaña-Pérez, V; Navarro-González, J F

    2014-01-01

    Paricalcitol, a selective vitamin D receptor (VDR) activator used for treatment of secondary hyperparathyroidism in chronic kidney disease (CKD), has been associated with survival advantages, suggesting that this drug, beyond its ability to suppress parathyroid hormone, may have additional beneficial actions. In this prospective, nonrandomised, open-label, proof-of-concept study, we evaluated the hypothesis that selective vitamin D receptor activation with paricalcitol is an effective target to modulate inflammation in CKD patients. Eight patients with an estimated glomerular filtration rate between 15 and 44?mL/min/1.73?m(2) and an intact parathyroid hormone (PTH) level higher than 110?pg/mL received oral paricalcitol (1? ?g/48 hours) as therapy for secondary hyperparathyroidism. Nine patients matched by age, sex, and stage of CKD, but a PTH level <110?pg/mL, were enrolled as a control group. Our results show that five months of paricalcitol administration were associated with a reduction in serum concentrations of hs-CRP (13.9%, P < 0.01), TNF-? (11.9%, P = 0.01), and IL-6 (7%, P < 0.05), with a nonsignificant increase of IL-10 by 16%. In addition, mRNA expression levels of the TNF? and IL-6 genes in peripheral blood mononuclear cells decreased significantly by 30.8% (P = 0.01) and 35.4% (P = 0.01), respectively. In conclusion, selective VDR activation is an effective target to modulate inflammation in CKD. PMID:24511210

  5. Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses.

    PubMed

    Hildebrand, Laura; Seemann, Petra; Kurtz, Andreas; Hecht, Jochen; Contzen, Jörg; Gossen, Manfred; Stachelscheid, Harald

    2015-12-01

    Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA(+) cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSC(TVA+) as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA(+) cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies. PMID:26109426

  6. Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma.

    PubMed

    Bogen, Dominik; Wei, Jun S; Azorsa, David O; Ormanoglu, Pinar; Buehler, Eugen; Guha, Rajarshi; Keller, Jonathan M; Mathews Griner, Lesley A; Ferrer, Marc; Song, Young K; Liao, Hongling; Mendoza, Arnulfo; Gryder, Berkley E; Sindri, Sivasish; He, Jianbin; Wen, Xinyu; Zhang, Shile; Shern, John F; Yohe, Marielle E; Taschner-Mandl, Sabine; Shohet, Jason M; Thomas, Craig J; Martin, Scott E; Ambros, Peter F; Khan, Javed

    2015-11-01

    Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. PMID:26497213

  7. Host factors in retroviral integration and the selection of integration target sites

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2015-01-01

    In order to replicate, a retrovirus must integrate a DNA copy of the viral RNA genome into a chromosome of the host cell. The study of retroviral integration has advanced considerably in the last few years. Here we focus on host factor interactions and the linked area of integration targeting. Genome-wide screens for cellular factors affecting HIV replication have identified a series of host cell proteins that may mediate subcellular trafficking of integration complexes, nuclear import, and integration target site selection. The cell transcriptional co-activator protein LEDGF/p75 has been identified as a tethering factor important for HIV integration, and recently, BET proteins (Brd2, 4, and 4) have been identified as tethering factors for the gammaretroviruses. A new class of HIV inhibitors has been developed targeting the HIV-1 IN-LEDGF binding site, though surprisingly these inhibitors appear to block assembly late during replication and do not act at the integration step. Going forward, genome-wide studies of HIV-host interactions offer many new starting points to investigate HIV replication and identify potential new inhibitor targets. PMID:26104434

  8. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  9. Apoptosis related protein 3 is a lysosomal membrane protein.

    PubMed

    Ding, XiaoDong; Chen, YuanWen; Han, LianShu; Qiu, WenJuan; Gu, XueFan; Zhang, HuiWen

    2015-05-15

    Apoptosis Related Protein 3 (APR3) is an important protein which is involved in retinoic acid-induced apoptosis, osteoblast differentiation and cervical squamous cell carcinoma progression. Although it was predicted to be a trans-membrane protein, its cellular localization is not clear. In this study, we analyzed APR3 with bioinformatic tools and found that APR3 contains a potential signal peptide, a transmembrane region and 3 N-glycosylation sites, all of which are characteristics of lysosomal proteins. Western blot with isolated lysosomes demonstrated that APR3 was mainly present in lysosomes, specially in the lysosomal membrane fraction, but not in endoplasmic reticulum. Concomitantly, double immunofluorescence confirmed that APR3 co-localized with lysosomal membrane protein, LAMP1, as well as lysosomal specific marker, Lyso-Tracker Red. Moreover, we showed that APR3 was highly expressed in the lung, liver, spleen, kidney and adipose tissue, but expressed at the low level in the heart, pancreas, stomach and intestine. Interestingly, APR3 expression was elevated in multiple hepatocellular carcinoma cell lines comparing to normal liver cells. Collectively, our results proved that APR3 is a novel lysosomal membrane protein and shed light on its possible functions. PMID:25839652

  10. Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry

    PubMed Central

    Zhang, X. Kate; Elbin, Carole S.; Turecek, Frantisek; Scott, Ronald; Chuang, Wei-Lien; Keutzer, Joan M.; Gelb, Michael

    2012-01-01

    Deficiencies in any of the 50 degradative enzymes found in lysosomes results in the accumulation of undegraded material and subsequently cellular dysfunction. Early identification of deficiencies before irreversible organ and tissue damages occur leads to better clinical outcomes. In the method which follows, lysosomal ?-glucosidase, ?-galactosidase, ?-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase are extracted from dried blood spots and incubated individually with an enzyme-specific cocktail containing the corresponding substrate and internal standard. Each enzyme cocktail is prepared using commercially available mixture of substrate and internal standard at the predetermined optimized molar ratio. After incubation, the enzymatic reactions are quenched using an ethyl acetate/methanol solution and all five enzyme solutions are combined. The mixtures of the reaction products are prepared using liquid–liquid and solid-phase extractions and quantified simultaneously using selected ion monitoring on LC-MS-MS system. PMID:20077085

  11. Target selection and mass estimation for manned NEO exploration using a baseline mission design

    NASA Astrophysics Data System (ADS)

    Boden, Ralf C.; Hein, Andreas M.; Kawaguchi, Junichiro

    2015-06-01

    In recent years Near-Earth Objects (NEOs) have received an increased amount of interest as a target for human exploration. NEOs offer scientifically interesting targets, and at the same time function as a stepping stone for achieving future Mars missions. The aim of this research is to identify promising targets from the large number of known NEOs that qualify for a manned sample-return mission with a maximum duration of one year. By developing a baseline mission design and a mass estimation model, mission opportunities are evaluated based on on-orbit mass requirements, safety considerations, and the properties of the potential targets. A selection of promising NEOs is presented and the effects of mission requirements and restrictions are discussed. Regarding safety aspects, the use of free-return trajectories provides the lowest on-orbit mass, when compared to an alternative design that uses system redundancies to ensure return of the spacecraft to Earth. It is discovered that, although a number of targets are accessible within the analysed time frame, no NEO offers both easy access and high incentive for its exploration. Under the discussed aspects a first human exploration mission going beyond the vicinity of Earth will require a trade off between targets that provide easy access and those that are of scientific interest. This lack of optimal mission opportunities can be seen in the small number of only 4 NEOs that meet all requirements for a sample-return mission and remain below an on-orbit mass of 500 metric Tons (mT). All of them require a mass between 315 and 492 mT. Even less ideal, smaller asteroids that are better accessible require an on-orbit mass that exceeds the launch capability of future heavy lift vehicles (HLV) such as SLS by at least 30 mT. These mass requirements show that additional efforts are necessary to increase the number of available targets and reduce on-orbit mass requirements through advanced mission architectures. The need for on-orbit assembly also becomes apparent, as availability of a HLV alone does not provide sufficient payload capabilities for any manned mission targeting NEOs.

  12. Rapid and targeted introgression of genes into popular wheat cultivars using marker-assisted background selection.

    PubMed

    Randhawa, Harpinder S; Mutti, Jasdeep S; Kidwell, Kim; Morris, Craig F; Chen, Xianming; Gill, Kulvinder S

    2009-01-01

    A marker-assisted background selection (MABS)-based gene introgression approach in wheat (Triticum aestivum L.) was optimized, where 97% or more of a recurrent parent genome (RPG) can be recovered in just two backcross (BC) generations. A four-step MABS method was developed based on 'Plabsim' computer simulations and wheat genome structure information. During empirical optimization of this method, double recombinants around the target gene were selected in a step-wise fashion during the two BC cycles followed by selection for recurrent parent genotype on non-carrier chromosomes. The average spacing between carrier chromosome markers was <4 cM. For non-carrier chromosome markers that flanked each of the 48 wheat gene-rich regions, this distance was approximately 12 cM. Employed to introgress seedling stripe rust (Puccinia striiformis f. sp. tritici) resistance gene Yr15 into the spring wheat cultivar 'Zak', marker analysis of 2,187 backcross-derived progeny resulted in the recovery of a BC(2)F(2ratio3) plant with 97% of the recurrent parent genome. In contrast, only 82% of the recurrent parent genome was recovered in phenotypically selected BC(4)F(7) plants developed without MABS. Field evaluation results from 17 locations indicated that the MABS-derived line was either equal or superior to the recurrent parent for the tested agronomic characteristics. Based on these results, MABS is recommended as a strategy for rapidly introgressing a targeted gene into a wheat genotype in just two backcross generations while recovering 97% or more of the recurrent parent genotype. PMID:19484121

  13. The SDSS-IV Extended Baryon Oscillation Spectroscopic Survey: Quasar Target Selection

    NASA Astrophysics Data System (ADS)

    Myers, Adam D.; Palanque-Delabrouille, Nathalie; Prakash, Abhishek; Pâris, Isabelle; Yeche, Christophe; Dawson, Kyle S.; Bovy, Jo; Lang, Dustin; Schlegel, David J.; Newman, Jeffrey A.; Petitjean, Patrick; Kneib, Jean-Paul; Laurent, Pierre; Percival, Will J.; Ross, Ashley J.; Seo, Hee-Jong; Tinker, Jeremy L.; Armengaud, Eric; Brownstein, Joel; Burtin, Etienne; Cai, Zheng; Comparat, Johan; Kasliwal, Mansi; Kulkarni, Shrinivas R.; Laher, Russ; Levitan, David; McBride, Cameron K.; McGreer, Ian D.; Miller, Adam A.; Nugent, Peter; Ofek, Eran; Rossi, Graziano; Ruan, John; Schneider, Donald P.; Sesar, Branimir; Streblyanska, Alina; Surace, Jason

    2015-12-01

    As part of the Sloan Digital Sky Survey (SDSS) IV the extended Baryon Oscillation Spectroscopic Survey (eBOSS) will improve measurements of the cosmological distance scale by applying the Baryon Acoustic Oscillation (BAO) method to quasar samples. eBOSS will adopt two approaches to target quasars over 7500 deg2. First, a “CORE” quasar sample will combine the optical selection in ugriz using a likelihood-based routine called XDQSOz, with a mid-IR-optical color cut. eBOSS CORE selection (to g < 22 or r < 22) should return ?70 deg?2 quasars at redshifts 0.9 < z < 2.2 and ?7 deg?2z > 2.1 quasars. Second, a selection based on variability in multi-epoch imaging from the Palomar Transient Factory should recover an additional ?3–4 deg?2z > 2.1 quasars to g < 22.5. A linear model of how imaging systematics affect target density recovers the angular distribution of eBOSS CORE quasars over 96.7% (76.7%) of the SDSS north (south) Galactic Cap area. The eBOSS CORE quasar sample should thus be sufficiently dense and homogeneous over 0.9 < z < 2.2 to yield the first few-percent-level BAO constraint near \\bar{z}? 1.5.eBOSS quasars at z > 2.1 will be used to improve BAO measurements in the Ly? Forest. Beyond its key cosmological goals, eBOSS should be the next-generation quasar survey, comprising >500,000 new quasars and >500,000 uniformly selected spectroscopically confirmed 0.9 < z < 2.2 quasars. At the conclusion of eBOSS, the SDSS will have provided unique spectra for more than 800,000 quasars.

  14. The diffusion properties of ion implanted species in selected target materials

    SciTech Connect

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

    1995-02-01

    Experiments important to the future success of the Holifield Radioactive Ion Beam Facility (HRIBF) are in progress at the Oak Ridge National Laboratory which are designed to select the most appropriate target material for generating a particular radioactive ion beam (RIB). The 25-MV HHIRF tandem accelerator is used to implant stable complements of interesting radioactive elements into refractory targets mounted in a high-temperature FEBIAD ion source which is {open_quotes}on-line{close_quotes} at the UNISOR facility. The intensity versus time of implanted species, which diffuse from the high-temperature target material ({approximately}1700{degrees}C) and are ionized in the FEBIAD ion source, is used to determine release times for a particular projectile/target material combination. From such release data, diffusion coefficients can be derived by fitting the theoretical results obtained by computational solution of Fick`s second equation to experimental data. The diffusion coefficient can be used subsequently to predict the release properties of the particular element from the same material in other target geometries and at other temperatures, provided that the activation energy is also known. Diffusion coefficients for Cl implanted into and diffused from CeS and Zr{sub 5}Si{sub 3} and As, Br, and Se implanted into and diffused from Zr{sub 5}Ge{sub 3} have been derived from the resulting intensity versus time profiles. Brief descriptions of the experimental apparatus and procedures utilized in the present experiments and plans for future related experiments are presented.

  15. Targeted Prostate Biopsy to Select Men for Active Surveillance—Do the Epstein Criteria Still Apply?

    PubMed Central

    Hu, Jim C.; Chang, Edward; Natarajan, Shyam; Margolis, Daniel J.; Macairan, Malu; Lieu, Patricia; Huang, Jiaoti; Sonn, Geoffrey; Dorey, Frederick J.; Marks, Leonard S.

    2014-01-01

    Purpose Established in 1994, the Epstein histological criteria (Gleason score 6 or less, 2 or fewer cores positive and 50% or less of any core) have been widely used to select men for active surveillance. However, with the advent of targeted biopsy, which may be more accurate than conventional biopsy, we reevaluated the likelihood of reclassification upon confirmatory rebiopsy using multiparametric magnetic resonance imaging-ultrasound fusion. Materials and Methods We identified 113 men enrolled in active surveillance at our institution who met Epstein criteria and subsequently underwent confirmatory targeted biopsy via multiparametric magnetic resonance imaging-ultrasound fusion. Median patient age was 64 years, median prostate specific antigen was 4.2 ng/ml and median prostate volume was 46.8 cc. Targets or regions of interest on multiparametric magnetic resonance imaging-ultrasound fusion were graded by suspicion level and biopsied at 3 mm intervals along the longest axis (median 10.5 mm). Also, 12 systematic cores were obtained during confirmatory rebiopsy. Our reporting is consistent with START (Standards of Reporting for MRI-targeted Biopsy Studies) criteria. Results Confirmatory fusion biopsy resulted in reclassification in 41 men (36%), including 26 (23%) due to Gleason grade 6 or greater and 15 (13%) due to high volume Gleason 6 disease. When stratified by suspicion on multiparametric magnetic resonance imaging-ultrasound fusion, the likelihood of reclassification was 24% to 29% for target grade 0 to 3, 45% for grade 4 and 100% for grade 5 (p = 0.001). Men with grade 4 and 5 vs lower grade targets were greater than 3 times more likely to be reclassified (OR 3.2, 95% CI 1.4–7.1, p = 0.006). Conclusions Upon confirmatory rebiopsy using multiparametric magnetic resonance imaging-ultrasound fusion men with high suspicion targets on imaging were reclassified 45% to 100% of the time. Criteria for active surveillance should be reevaluated when multiparametric magnetic resonance imaging-ultrasound fusion guided prostate biopsy is used. PMID:24512956

  16. Status of autophagy, lysosome activity and apoptosis during corpus luteum regression in cattle

    PubMed Central

    ABOELENAIN, Mansour; KAWAHARA, Manabu; BALBOULA, Ahmed Zaky; MONTASSER, Abd El-monem; ZAABEL, Samy Mowaed; OKUDA, Kiyoshi; TAKAHASHI, Masashi

    2015-01-01

    Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3?, LC3?, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL. PMID:25819401

  17. The Deubiquitinating Enzyme UBPY Is Required for Lysosomal Biogenesis and Productive Autophagy in Drosophila

    PubMed Central

    Jacomin, Anne-Claire; Bescond, Amandine; Soleilhac, Emmanuelle; Gallet, Benoît; Schoehn, Guy; Fauvarque, Marie-Odile; Taillebourg, Emmanuel

    2015-01-01

    Autophagy is a catabolic process that delivers cytoplasmic components to the lysosomes. Protein modification by ubiquitination is involved in this pathway: it regulates the stability of autophagy regulators such as BECLIN-1 and it also functions as a tag targeting specific substrates to autophagosomes. In order to identify deubiquitinating enzymes (DUBs) involved in autophagy, we have performed a genetic screen in the Drosophila larval fat body. This screen identified Uch-L3, Usp45, Usp12 and Ubpy. In this paper, we show that Ubpy loss of function results in the accumulation of autophagosomes due to a blockade of the autophagy flux. Furthermore, analysis by electron and confocal microscopy of Ubpy-depleted fat body cells revealed altered lysosomal morphology, indicating that Ubpy inactivation affects lysosomal maintenance and/or biogenesis. Lastly, we have shown that shRNA mediated inactivation of UBPY in HeLa cells affects autophagy in a different way: in UBPY-depleted HeLa cells autophagy is deregulated. PMID:26571504

  18. Speed-Selectivity Paradox in the Protein Search for Targets on DNA: Is It Real or Not?

    E-print Network

    Speed-Selectivity Paradox in the Protein Search for Targets on DNA: Is It Real or Not? Alex Veksler and Anatoly B. Kolomeisky* Department of Chemistry, Rice University, Houston, Texas 77005, United States ABSTRACT: Protein search for targets on DNA starts all major biological processes. Although significant

  19. 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

  20. 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.

  1. 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.

  2. Does Angling Technique Selectively Target Fishes Based on Their Behavioural Type?

    PubMed Central

    Wilson, Alexander D. M.; Brownscombe, Jacob W.; Sullivan, Brittany; Jain-Schlaepfer, Sofia; Cooke, Steven J.

    2015-01-01

    Recently, there has been growing recognition that fish harvesting practices can have important impacts on the phenotypic distributions and diversity of natural populations through a phenomenon known as fisheries-induced evolution. Here we experimentally show that two common recreational angling techniques (active crank baits versus passive soft plastics) differentially target wild largemouth bass (Micropterus salmoides) and rock bass (Ambloplites rupestris) based on variation in their behavioural tendencies. Fish were first angled in the wild using both techniques and then brought back to the laboratory and tested for individual-level differences in common estimates of personality (refuge emergence, flight-initiation-distance, latency-to-recapture and with a net, and general activity) in an in-lake experimental arena. We found that different angling techniques appear to selectively target these species based on their boldness (as characterized by refuge emergence, a standard measure of boldness in fishes) but not other assays of personality. We also observed that body size was independently a significant predictor of personality in both species, though this varied between traits and species. Our results suggest a context-dependency for vulnerability to capture relative to behaviour in these fish species. Ascertaining the selective pressures angling practices exert on natural populations is an important area of fisheries research with significant implications for ecology, evolution, and resource management. PMID:26284779

  3. Selective Janus associated kinase 1 inhibition as a therapeutic target in myelofibrosis.

    PubMed

    Mascarenhas, John

    2015-09-01

    Hyperactive Janus associated kinase-signal transducers and activators of transcription (JAK-STAT) signaling has been shown to be integral to the pathogenesis of myelofibrosis (MF) regardless of the driver mutational status (JAK2V617F, JAK2 exon 12, MPL515L/K, CALR). Targeting of the JAK-STAT pathway has been the intense focus of therapeutic development and led to the approval of the JAK1/2 inhibitor, ruxolitinib. Despite the clear clinical success of ruxolitinib, dose limiting thrombocytopenia, treatment associated anemia and failure to effectively achieve bone marrow pathologic, cytogenetic and molecular remission remain shortcomings. JAK1 inhibition leads to depression in inflammatory cytokine expression associated with MF-related constitutional symptoms. The selective targeting of JAK1 may provide an opportunity to alleviate MF-related symptoms without anti-JAK2 therapy-related myelosuppression. Additionally, a JAK1 inhibitor may serve as an ideal candidate partner for combination therapeutic approaches in the treatment of MF. Current evaluation of selective JAK1 inhibition in MF will further clarify the relative contribution of aberrant JAK1 signaling to the pathogenesis of MF. PMID:25586607

  4. Selective targeting of bioengineered platelets to prostate cancer vasculature: new paradigm for therapeutic modalities

    PubMed Central

    Montecinos, Viviana P; Morales, Claudio H; Fischer, Thomas H; Burns, Sarah; San Francisco, Ignacio F; Godoy, Alejandro S; Smith, Gary J

    2015-01-01

    Androgen deprivation therapy (ADT) provides palliation for most patients with advanced prostate cancer (CaP); however, greater than 80% subsequently fail ADT. ADT has been indicated to induce an acute but transient destabilization of the prostate vasculature in animal models and humans. Human re-hydrated lyophilized platelets (hRL-P) were investigated as a prototype for therapeutic agents designed to target selectively the tumour-associated vasculature in CaP. The ability of hRL-P to bind the perturbed endothelial cells was tested using thrombin- and ADP-activated human umbilical vein endothelial cells (HUVEC), as well as primary xenografts of human prostate tissue undergoing acute vascular involution in response to ADT. hRL-P adhered to activated HUVEC in a dose-responsive manner. Systemically administered hRL-P, and hRL-P loaded with super-paramagnetic iron oxide (SPIO) nanoparticles, selectively targeted the ADT-damaged human microvasculature in primary xenografts of human prostate tissue. This study demonstrated that hRL-P pre-loaded with chemo-therapeutics or nanoparticles could provide a new paradigm for therapeutic modalities to prevent the rebound/increase in prostate vasculature after ADT, inhibiting the transition to castration-recurrent growth. PMID:25736582

  5. 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.

  6. Characterizing the pocketome of Mycobacterium tuberculosis and application in rationalizing polypharmacological target selection.

    PubMed

    Anand, Praveen; Chandra, Nagasuma

    2014-01-01

    Polypharmacology is beginning to emerge as an important concept in the field of drug discovery. However, there are no established approaches to either select appropriate target sets or design polypharmacological drugs. Here, we propose a structural-proteomics approach that utilizes the structural information of the binding sites at a genome-scale obtained through in-house algorithms to characterize the pocketome, yielding a list of ligands that can participate in various biochemical events in the mycobacterial cell. The pocket-type space is seen to be much larger than the sequence or fold-space, suggesting that variations at the site-level contribute significantly to functional repertoire of the organism. All-pair comparisons of binding sites within Mycobacterium tuberculosis (Mtb), pocket-similarity network construction and clustering result in identification of binding-site sets, each containing a group of similar binding sites, theoretically having a potential to interact with a common set of compounds. A polypharmacology index is formulated to rank targets by incorporating a measure of druggability and similarity to other pockets within the proteome. This study presents a rational approach to identify targets with polypharmacological potential along with possible drugs for repurposing, while simultaneously, obtaining clues on lead compounds for use in new drug-discovery pipelines. PMID:25220818

  7. 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.

  8. Asteroid target selection for the new Rosetta mission baseline. 21 Lutetia and 2867 Steins

    NASA Astrophysics Data System (ADS)

    Barucci, M. A.; Fulchignoni, M.; Fornasier, S.; Dotto, E.; Vernazza, P.; Birlan, M.; Binzel, R. P.; Carvano, J.; Merlin, F.; Barbieri, C.; Belskaya, I.

    2005-01-01

    The new Rosetta mission baseline to the comet 67P/Churyumov-Gerasimenko includes two asteroid fly-bys. To help in target selection we studied all the candidates of all the possible scenarios. Observations have been carried out at ESO-NTT (La Silla, Chile), TNG (Canaries), and NASA-IRTF (Hawaii) telescopes, in order to determine the taxonomy of all the candidates. The asteroid targets were chosen after the spacecraft interplanetary orbit insertion manoeuvre, when the available total amount of ? V was known. On the basis of our analysis and the available of ? V, we recommended to the ESA Science Working Group the asteroids 21 Lutetia and 2867 Steins as targets for the Rosetta mission. The nature of Lutetia is still controversial. Lutetia's spectral properties may be consistent with a composition similar to carbonaceous chondrite meteorites. The spectral properties of Steins suggest a more extensive thermal history. Steins may have a composition similar to relatively rare enstatite chondrite/achondrite meteorites. Based on observations collected at ESO-La Silla, NASA/IRTF and TNG-Canaries.

  9. 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 ...

  10. 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

  11. Selective inhibition of the unfolded protein response: targeting catalytic sites for Schiff base modification.

    PubMed

    Tomasio, Susana M; Harding, Heather P; Ron, David; Cross, Benedict C S; Bond, Peter J

    2013-10-01

    Constitutive protein misfolding in the endoplasmic reticulum (ER) can lead to cellular toxicity and disease. Consequently, the protein folding environment within the ER is highly optimised and tightly regulated by the unfolded protein response (UPR). The apparent convergence of myriad diseases upon proteostasis in the ER has triggered a broad effort to identify selective inhibitors of the UPR. In particular, the most ancient component of this cellular stress pathway, the transmembrane protein IRE1, represents an appealing target for pharmacological intervention. Several inhibitors of IRE1 have recently been reported, each containing an aldehyde moiety that forms an unusual, highly selective Schiff base with a single key lysine (K907) within the RNase domain. Here we review the progress made in chemical genetic manipulation of IRE1 and the unfolded protein response and discuss computational strategies to rationalise the selectivity of covalently active small molecules for their targets. As an exemplar, we provide additional evidence that K907 of IRE1 is buried within a particularly unusual environment that facilitates Schiff base formation. New free-energy calculations within a molecular dynamics (MD) simulation framework show that the pKa of K907 is reduced by ~3.6 pKa units, relative to the model pKa of lysine in water. This significant pKa perturbation provides additional insights into the precise requirements for inhibition and for RNase catalysis by IRE1. Our computational method may represent a general approach for identifying potential covalent inhibitory lysine sites within buried protein cavities. PMID:23884086

  12. Selection Against Maternal microRNA Target Sites in Maternal Transcripts

    PubMed Central

    Marco, Antonio

    2015-01-01

    In animals, before the zygotic genome is expressed, the egg already contains gene products deposited by the mother. These maternal products are crucial during the initial steps of development. In Drosophila melanogaster, a large number of maternal products are found in the oocyte, some of which are indispensable. Many of these products are RNA molecules, such as gene transcripts and ribosomal RNAs. Recently, microRNAs (small RNA gene regulators) have been detected early during development and are important in these initial steps. The presence of some microRNAs in unfertilized eggs has been reported, but whether they have a functional impact in the egg or early embryo has not being explored. I have extracted and sequenced small RNAs from Drosophila unfertilized eggs. The unfertilized egg is rich in small RNAs and contains multiple microRNA products. Maternal microRNAs often are encoded within the intron of maternal genes, suggesting that many maternal microRNAs are the product of transcriptional hitchhiking. Comparative genomics analyses suggest that maternal transcripts tend to avoid target sites for maternal microRNAs. I also developed a microRNA target mutation model to study the functional impact of polymorphisms at microRNA target sites. The analysis of Drosophila populations suggests that there is selection against maternal microRNA target sites in maternal transcripts. A potential role of the maternal microRNA mir-9c in maternal-to-zygotic transition is also discussed. In conclusion, maternal microRNAs in Drosophila have a functional impact in maternal protein?coding transcripts. PMID:26306531

  13. The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice.

    PubMed

    Alves, Sandro; Cormier-Dequaire, Florence; Marinello, Martina; Marais, Thibaut; Muriel, Marie-Paule; Beaumatin, Florian; Charbonnier-Beaupel, Fanny; Tahiri, Khadija; Seilhean, Danielle; El Hachimi, Khalid; Ruberg, Merle; Stevanin, Giovanni; Barkats, Martine; den Dunnen, Wilfred; Priault, Muriel; Brice, Alexis; Durr, Alexandra; Corvol, Jean-Christophe; Sittler, Annie

    2014-11-01

    There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-D were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene ATG12 was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders. PMID:24859968

  14. Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat.

    PubMed

    Robb, Ellen L; Gawel, Justyna M; Aksentijevi?, Dunja; Cochemé, Helena M; Stewart, Tessa S; Shchepinova, Maria M; Qiang, He; Prime, Tracy A; Bright, Thomas P; James, Andrew M; Shattock, Michael J; Senn, Hans M; Hartley, Richard C; Murphy, Michael P

    2015-12-01

    Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo. PMID:26454075

  15. Characterization of an oligopeptide transporter in renal lysosomes.

    PubMed

    Zhou, X; Thamotharan, M; Gangopadhyay, A; Serdikoff, C; Adibi, S A

    2000-06-01

    Renal lysosomes play a major role in catabolism of plasma proteins. Final products of this catabolism include dipeptides and tripeptides that must be exported to the cytosol for hydrolysis. The aim of the present study was to determine whether an oligopeptide transporter is present in the renal lysosomal membrane that could mediate this export. The existence of an oligopeptide transporter was probed with the uptake of glycylglutamine (Gly-Gln) by membrane vesicles prepared from renal lysosomes. Kinetic analysis showed the presence of a single transporter with a K(m) of 8.77 mM for the uptake of Gly-Gln. The Gly-Gln uptake was energized by the imposition of an inwardly directed proton gradient (pH(out) 5.0/pH(in) 7.3) and membrane potential (outside positive/inside negative) resulting in overshoot. The Gly-Gln uptake was inhibited by the presence of dipeptides and tripeptides, but not amino acids. Western blot analysis of lysosomal membrane proteins with Pept-1 (an oligopeptide transporter) antibody as the probe showed the presence of an immunoreactive protein. This immunoreaction was abolished when the antiserum was preabsorbed with the Pept-1 epitope (0.5 microg/ml). In conclusion, the present data show the existence of a low-affinity dipeptide transporter in the renal lysosomal membrane that appears to belong to the Pept family of transporters. The function of this transporter appears to be to prevent accumulation of dipeptides in renal lysosomes. PMID:10825457

  16. RTB Lectin: a novel receptor-independent delivery system for lysosomal enzyme replacement therapies

    PubMed Central

    Acosta, Walter; Ayala, Jorge; Dolan, Maureen C.; Cramer, Carole L.

    2015-01-01

    Enzyme replacement therapies have revolutionized patient treatment for multiple rare lysosomal storage diseases but show limited effectiveness for addressing pathologies in “hard-to-treat” organs and tissues including brain and bone. Here we investigate the plant lectin RTB as a novel carrier for human lysosomal enzymes. RTB enters mammalian cells by multiple mechanisms including both adsorptive-mediated and receptor-mediated endocytosis, and thus provides access to a broader array of organs and cells. Fusion proteins comprised of RTB and human ?-L-iduronidase, the corrective enzyme for Mucopolysaccharidosis type I, were produced using a tobacco-based expression system. Fusion products retained both lectin selectivity and enzyme activity, were efficiently endocytosed into human fibroblasts, and corrected the disease phenotype of mucopolysaccharidosis patient fibroblasts in vitro. RTB-mediated delivery was independent of high-mannose and mannose-6-phosphate receptors, which are exploited for delivery of currently approved lysosomal enzyme therapeutics. Thus, the RTB carrier may support distinct in vivo pharmacodynamics with potential to address hard-to-treat tissues. PMID:26382970

  17. Studies on the involvement of lysosomes in estrogen action, I. Isolation and enzymatic properties of pig endometrial lysosomes.

    PubMed

    Sierralta, W; Truitt, A J; Jungblut, P W

    1978-04-01

    Pig endometrium cells, collected by curettage and homogenized in an all-glass Potter Elvehjem homogenizer, gave a considerably higher yield of intact mitochondria and lysosomes than homogenates of whole uterus obtained with the Ultraturrax or the Parr bomb. After homogenization of the cells and subfractionation in the presence of Mg2, mitochondria and lysosomes equilibrated at the same modal density in isopycnic centrifugation. Homogenization and subfractionation in buffers devoid of divalent cations and containing EDTA resulted in a decrease in the buoyant density of mitochondria, allowing for a separation from lysosomes. The pH optima and the specific activities of two mitochondrial enzymes and eight hydrolyases used as marker enzymes were determined. The morphological characteristics of fractions were established by electron microscopy. Preliminary results indicate an involvement of lysosomes in steroid metabolism rather than in steroid and receptor translocation into the nucleus. PMID:25838

  18. Selection between Michaelis–Menten and target-mediated drug disposition pharmacokinetic models

    PubMed Central

    Yan, Xiaoyu; Mager, Donald E.

    2011-01-01

    Target-mediated drug disposition (TMDD) models have been applied to describe the pharmacokinetics of drugs whose distribution and/or clearance are affected by its target due to high binding affinity and limited capacity. The Michaelis–Menten (M–M) model has also been frequently used to describe the pharmacokinetics of such drugs. The purpose of this study is to investigate conditions for equivalence between M–M and TMDD pharmacokinetic models and provide guidelines for selection between these two approaches. Theoretical derivations were used to determine conditions under which M–M and TMDD pharmacokinetic models are equivalent. Computer simulations and model fitting were conducted to demonstrate these conditions. Typical M–M and TMDD profiles were simulated based on literature data for an anti-CD4 monoclonal antibody (TRX1) and phenytoin administered intravenously. Both models were fitted to data and goodness of fit criteria were evaluated for model selection. A case study of recombinant human erythropoietin was conducted to qualify results. A rapid binding TMDD model is equivalent to the M–M model if total target density Rtot is constant, and RtotKD/(KD + C)2 ? 1 where KD represents the dissociation constant and C is the free drug concentration. Under these conditions, M–M parameters are defined as: Vmax = kintRtotVc and Km = KD where kint represents an internalization rate constant, and Vc is the volume of the central compartment. Rtot is constant if and only if kint = kdeg, where kdeg is a degradation rate constant. If the TMDD model predictions are not sensitive to kint or kdeg parameters, the condition of RtotKD/(KD + C)2 ? 1 alone can preserve the equivalence between rapid binding TMDD and M–M models. The model selection process for drugs that exhibit TMDD should involve a full mechanistic model as well as reduced models. The best model should adequately describe the data and have a minimal set of parameters estimated with acceptable precision. PMID:20012173

  19. Selection between Michaelis-Menten and target-mediated drug disposition pharmacokinetic models.

    PubMed

    Yan, Xiaoyu; Mager, Donald E; Krzyzanski, Wojciech

    2010-02-01

    Target-mediated drug disposition (TMDD) models have been applied to describe the pharmacokinetics of drugs whose distribution and/or clearance are affected by its target due to high binding affinity and limited capacity. The Michaelis-Menten (M-M) model has also been frequently used to describe the pharmacokinetics of such drugs. The purpose of this study is to investigate conditions for equivalence between M-M and TMDD pharmacokinetic models and provide guidelines for selection between these two approaches. Theoretical derivations were used to determine conditions under which M-M and TMDD pharmacokinetic models are equivalent. Computer simulations and model fitting were conducted to demonstrate these conditions. Typical M-M and TMDD profiles were simulated based on literature data for an anti-CD4 monoclonal antibody (TRX1) and phenytoin administered intravenously. Both models were fitted to data and goodness of fit criteria were evaluated for model selection. A case study of recombinant human erythropoietin was conducted to qualify results. A rapid binding TMDD model is equivalent to the M-M model if total target density R ( tot ) is constant, and R ( tot ) K ( D ) /(K ( D ) + C) ( 2 ) < 1 where K ( D ) represents the dissociation constant and C is the free drug concentration. Under these conditions, M-M parameters are defined as: V ( max ) = k ( int ) R ( tot ) V ( c ) and K ( m ) = K ( D ) where k ( int ) represents an internalization rate constant, and V ( c ) is the volume of the central compartment. R ( tot ) is constant if and only if k ( int ) = k ( deg,) where k ( deg ) is a degradation rate constant. If the TMDD model predictions are not sensitive to k ( int ) or k ( deg ) parameters, the condition of R ( tot ) K ( D ) /(K ( D ) + C) ( 2 ) < 1 alone can preserve the equivalence between rapid binding TMDD and M-M models. The model selection process for drugs that exhibit TMDD should involve a full mechanistic model as well as reduced models. The best model should adequately describe the data and have a minimal set of parameters estimated with acceptable precision. PMID:20012173

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

    PubMed

    Guerrero-Preston, Rafael; Ogawa, Takenori; Uemura, Mamoru; Shumulinsky, Gary; Valle, Blanca L; Pirini, Francesca; Ravi, Rajani; Sidransky, David; Keidar, Michael; Trink, Barry

    2014-10-01

    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

  1. 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

  2. Pharmacological targeting of dopamine D3 receptors: Possible clinical applications of selective drugs.

    PubMed

    Pich, Emilio Merlo; Collo, Ginetta

    2015-09-01

    Dopamine D3 receptors have been pharmacologically engaged in humans since the development of the first antipsychotics and ergot-derivative dopamine (DA) agonists, even without knowing it. These agents were generally non-selective, developed primarily to target D2 receptors. In the last 10 years the understanding of the clinical implication of D3 receptors has been progressing also due to the identification of D3 gene polymorphisms, the use of more selective PET ligands such as [(11)C]-(+)-PHNO and the learning regarding the clinical use of the D3-preferential D2/D3 agonists ropinirole and pramipexole. A new specific neuroplasticity role of D3 receptor regarding dendrite arborisation outgrowth in dopaminergic neurons was also proposed to support, at least in part, the slowing of disease observed in subjects with Parkinson?s Disease treated with DA agonists. Similar mechanisms could be at the basis of the antidepressant-like effects observed with DA agonists when co-administered with standard of care. Severe adverse event occurring with the use of anti-parkinsonian DA agonists in predisposed subjects, i.e., impulse control disorders, are now suggested to be putatively related to overactive D3 receptors. Not surprisingly, blockade of D3 receptors was proposed as treatment for addictive disorders, a goal that could be potentially achieved by repositioning buspirone, an anxiolytic drug with D3-preferential antagonistic features, or with novel selective D3 antagonists or partial agonists currently in development for schizophrenia. At the moment ABT-925 is the only selective D3 antagonist tested in schizophrenic patients in Phase II, showing an intriguing cognitive enhancing effects supported by preclinical data. Finally, exploratory pharmacogenetic analysis suggested that ABT-925 could be effective in a subpopulation of patients with a polymorphism on the D3 receptor, opening to a possible personalised medicine approach. PMID:26298833

  3. New molecules and old drugs as emerging approaches to selectively target human glioblastoma cancer stem cells.

    PubMed

    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

  4. Tumor-targeted delivery of liposome-encapsulated doxorubicin by use of a peptide that selectively binds to irradiated tumors

    PubMed Central

    Lowery, Amanda; Onishko, Halina; Hallahan, Dennis E.; Han, Zhaozhong

    2010-01-01

    Tumor-targeted drug delivery improves anti-tumor efficacy and reduces systemic toxicity by limiting bioavailability of cytotoxic drugs to within tumors. Targeting reagents, such as peptides or antibodies recognizing molecular targets over-expressed within tumors, have been used to improve liposome-encapsulated drug accumulation within tumors and resulted in enhanced tumor growth control. In this report, we expand the scope of targeting reagents by showing that one peptide, HVGGSSV which was isolated from an in vivo screening of phage-displayed peptide library due to its selective binding within irradiated tumors, enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in enhanced cytotoxicity within tumors. Targeting liposomes (TL) and non-targeting liposomes (nTL) were labeled with Alexa Fluor 750. Biodistribution of the liposomes within tumor-bearing mice was studied with near infrared (NIR) imaging. In the single dose pharmacokinetic study, the liposomal doxorubicin has an extended circulation half life as compared to the free doxorubicin. Targeting liposomes partitioned to the irradiated tumors and improved drug deposition and retention within tumors. The tumor-targeted delivery of doxorubicin improved tumor growth control as indicated with reduced tumor growth rate and tumor cell proliferation, enhanced tumor blood vessel destruction, and increased treatment-associated apoptosis and necrosis of tumor cells. Collectively, the results demonstrated the remarkable capability of the HVGGSSV peptide in radiation-guided drug delivery to tumors. PMID:21075152

  5. Clinical Features of Lysosomal Acid Lipase Deficiency

    PubMed Central

    Burton, Barbara K.; Deegan, Patrick B.; Enns, Gregory M.; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K.; Lobritto, Steve J.; Malinova, Vera; McLin, Valerie A.; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B.; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G.

    2015-01-01

    Abstract Objective: The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Methods: Investigators reviewed medical records of LAL D patients ages ?5 years, extracted historical data, and obtained prospective laboratory and imaging data on living patients to develop a longitudinal dataset. Results: A total of 49 patients were enrolled; 48 had confirmed LAL D. Mean age at first disease-related abnormality was 9.0 years (range 0–42); mean age at diagnosis was 15.2 years (range 1–46). Twenty-nine (60%) were male patients, and 27 (56%) were <20 years of age at the time of consent/assent. Serum transaminases were elevated in most patients with 458 of 499 (92%) of alanine aminotransferase values and 265 of 448 (59%) of aspartate aminotransferase values above the upper limit of normal. Most patients had elevated low-density lipoprotein (64% patients) and total cholesterol (63%) at baseline despite most being on lipid-lowering therapies, and 44% had high-density lipoprotein levels below the lower limit of normal. More than half of the patients with liver biopsies (n?=?31, mean age 13 years) had documented evidence of steatosis (87%) and/or fibrosis (52%). Imaging assessments revealed that the median liver volume was ?1.15 multiples of normal (MN) and median spleen volume was ?2.2 MN. Six (13%) patients had undergone a liver transplant (ages 9–43.5 years). Conclusion: This study provides the largest longitudinal case review of patients with LAL D and confirms that LAL D is predominantly a pediatric disease causing early and progressive hepatic dysfunction associated with dyslipidemia that often leads to liver failure and transplantation. PMID:26252914

  6. Asteroid Target Selection and Orbital Manipulation Sensitivity for Deflection Demonstration Missions

    NASA Astrophysics Data System (ADS)

    Sanchez, J. P.

    2015-08-01

    In recent years, space agencies have begun to seriously consider launching demonstration missions to test some of the asteroid orbital deflection technologies and methods that have been studied and discussed in the scientific literature. Consequently, several mission studies have already been carried out. This paper attempts to gain new insights into the target selection process by analyzing the orbital evolution of a large set of notional accessible asteroids that cover all types of Near Earth Object families. The evolution of their unperturbed orbits and their anthropogenically modified trajectories was compared, and a measure of the resilience of a given orbit to anthropogenic manipulation was taken (i.e., orbital innocuity). The results show that pruning criteria such as considering only Amor objects (i.e., non-Earth-crossers) reduce unnecessarily the population of potential suitable targets and that within large regions of Earth-crossing orbital space asteroids can be found that are both accessible and safe to manipulate from the standpoint of the Earth impact risk.

  7. Selective Targeting of the TPX2 Site of Importin-? Using Fragment-Based Ligand Design

    PubMed Central

    Holvey, Rhian S; Valkov, Eugene; Neal, David; Stewart, Murray; Abell, Chris

    2015-01-01

    Protein–protein interactions are difficult therapeutic targets, and inhibiting pathologically relevant interactions without disrupting other essential ones presents an additional challenge. Herein we report how this might be achieved for the potential anticancer target, the TPX2–importin-? interaction. Importin-? is a nuclear transport protein that regulates the spindle assembly protein TPX2. It has two binding sites—major and minor—to which partners bind. Most nuclear transport cargoes use the major site, whereas TPX2 binds principally to the minor site. Fragment-based approaches were used to identify small molecules that bind importin-?, and crystallographic studies identified a lead series that was observed to bind specifically to the minor site, representing the first ligands specific for this site. Structure-guided synthesis informed the elaboration of these fragments to explore the source of ligand selectivity between the minor and major sites. These ligands are starting points for the development of inhibitors of this protein–protein interaction. PMID:25899172

  8. Targeting LSCs through membrane antigens selectively or preferentially expressed on these cells.

    PubMed

    Pelosi, Elvira; Castelli, Germana; Testa, Ugo

    2015-12-01

    Studies of xenotransplantation of bone marrow and blood cells of AML patients have supported the existence of rare leukemic stem cells, able to initiate and maintain the leukemic process and bearing the typical leukemic abnormalities. LSCs possess self-renewal capacity and are responsible for the growth of the more differentiated leukemic progeny in the bone marrow and in the blood. These cells are more resistant than bulk leukemic cells to anti-leukemic drugs, thus survive to treatment and are, at a large extent, responsible for leukemia relapse. During the last two decades, considerable progresses have been made in the understanding of the peculiar cellular and molecular properties of LSCs. In this context, particularly relevant was the discovery of several membrane markers, selectively or preferentially expressed on LSCs. These membrane markers offer now unique opportunities to identify LSCs and to distinguish them from normal HSCs, to monitor the response of the various anti-leukemic treatments at the level of the LSC compartment, to identify relevant therapeutic targets. Concerning this last point, the most promising therapeutic targets are CD33 and CD123. PMID:26460257

  9. Searching for life on Mars: selection of molecular targets for ESA's aurora ExoMars mission.

    PubMed

    Parnell, John; Cullen, David; Sims, Mark R; Bowden, Stephen; Cockell, Charles S; Court, Richard; Ehrenfreund, Pascale; Gaubert, Francois; Grant, William; Parro, Victor; Rohmer, Michel; Sephton, Mark; Stan-Lotter, Helga; Steele, Andrew; Toporski, Jan; Vago, Jorge

    2007-08-01

    The European Space Agency's ExoMars mission will seek evidence of organic compounds of biological and non-biological origin at the martian surface. One of the instruments in the Pasteur payload may be a Life Marker Chip that utilizes an immunoassay approach to detect specific organic molecules or classes of molecules. Therefore, it is necessary to define and prioritize specific molecular targets for antibody development. Target compounds have been selected to represent meteoritic input, fossil organic matter, extant (living, recently dead) organic matter, and contamination. Once organic molecules are detected on Mars, further information is likely to derive from the detailed distribution of compounds rather than from single molecular identification. This will include concentration gradients beneath the surface and gradients from generic to specific compounds. The choice of biomarkers is informed by terrestrial biology but is wide ranging, and nonterrestrial biology may be evident from unexpected molecular distributions. One of the most important requirements is to sample where irradiation and oxidation are minimized, either by drilling or by using naturally excavated exposures. Analyzing regolith samples will allow for the search of both extant and fossil biomarkers, but sequential extraction would be required to optimize the analysis of each of these in turn. PMID:17723091

  10. Phosphorylation of Liver X Receptor ? Selectively Regulates Target Gene Expression in Macrophages? †

    PubMed Central

    Torra, Inés Pineda; Ismaili, Naima; Feig, Jonathan E.; Xu, Chong-Feng; Cavasotto, Claudio; Pancratov, Raluca; Rogatsky, Inez; Neubert, Thomas A.; Fisher, Edward A.; Garabedian, Michael J.

    2008-01-01

    Dysregulation of liver X receptor ? (LXR?) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXR? target gene selectivity is achieved by modulation of LXR? phosphorylation. Under basal conditions, LXR? is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXR? S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXR? S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXR?-responsive genes. PMID:18250151

  11. 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

  12. Selective Targeting of the TPX2 Site of Importin-? Using Fragment-Based Ligand Design.

    PubMed

    Holvey, Rhian S; Valkov, Eugene; Neal, David; Stewart, Murray; Abell, Chris

    2015-07-01

    Protein-protein interactions are difficult therapeutic targets, and inhibiting pathologically relevant interactions without disrupting other essential ones presents an additional challenge. Herein we report how this might be achieved for the potential anticancer target, the TPX2-importin-? interaction. Importin-? is a nuclear transport protein that regulates the spindle assembly protein TPX2. It has two binding sites--major and minor-to which partners bind. Most nuclear transport cargoes use the major site, whereas TPX2 binds principally to the minor site. Fragment-based approaches were used to identify small molecules that bind importin-?, and crystallographic studies identified a lead series that was observed to bind specifically to the minor site, representing the first ligands specific for this site. Structure-guided synthesis informed the elaboration of these fragments to explore the source of ligand selectivity between the minor and major sites. These ligands are starting points for the development of inhibitors of this protein-protein interaction. PMID:25899172

  13. 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

  14. Poor lysosomal membrane integrity in proximal tubule cells of haptoglobin 2-2 genotype mice with diabetes mellitus

    PubMed Central

    Asleh, Rabea; Nakhoul, Farid M.; Miller-Lotan, Rachel; Awad, Hoda; Farbstein, Dan; Levy, Nina S.; Nakhoul, Nakhoul; Iancu, Theodore C.; Manov, Irena; Laue, Michael; Traber, Maret G.; Lebold, Katie M.; Levy, Andrew P.

    2013-01-01

    The haptoglobin (Hp) genotype is a major determinant of progression of nephropathy in individuals with diabetes mellitus (DM). The major function of the Hp protein is to bind and modulate the fate of extracorpuscular hemoglobin and its iron cargo. We have previously demonstrated an interaction between the Hp genotype and the DM on the accumulation of iron in renal proximal tubule cells. The primary objective of this study was to determine the intracellular localization of this iron in the proximal tubule cell and to assess its potential toxicity. Transmission electron microscopy demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubules cells in Hp 2-2 DM mice. Energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich. These deposits were associated with lysosomal membrane lipid peroxidation and loss of lysosomal membrane integrity. Vitamin E administration to Hp 2-2 DM mice resulted in a significant decrease in both intralysosomal iron-induced oxidation and lysosomal destabilization. Iron-induced renal tubular injury may play a major role in the development of diabetic nephropathy and may be a target for slowing the progression of renal disease. PMID:22749805

  15. A guide to picking the most selective kinase inhibitor tool compounds for pharmacological validation of drug targets

    PubMed Central

    Uitdehaag, Joost CM; Verkaar, Folkert; Alwan, Husam; de Man, Jos; Buijsman, Rogier C; Zaman, Guido JR

    2012-01-01

    To establish the druggability of a target, genetic validation needs to be supplemented with pharmacological validation. Pharmacological studies, especially in the kinase field, are hampered by the fact that many reference inhibitors are not fully selective for one target. Fortunately, the initial trickle of selective inhibitors released in the public domain has steadily swelled into a stream. However, rationally picking the most selective tool compound out of the increasing amounts of available inhibitors has become progressively difficult due to the lack of accurate quantitative descriptors of drug selectivity. A recently published approach, termed ‘selectivity entropy’, is an improved way of expressing selectivity as a single-value parameter and enables rank ordering of inhibitors. We provide a guide to select the best tool compounds for pharmacological validation experiments of candidate drug targets using selectivity entropy. In addition, we recommend which inhibitors to use for studying the biology of the 20 most investigated kinases that are clinically relevant: Abl (ABL1), AKT1, ALK, Aurora A/B, CDKs, MET, CSF1R (FMS), EGFR, FLT3, ERBB2 (HER2), IKBKB (IKK2), JAK2/3, JNK1/2/3 (MAPK8/9/10), MEK1/2, PLK1, PI3Ks, p38? (MAPK14), BRAF, SRC and VEGFR2 (KDR). PMID:22250956

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

    E-print Network

    Xiao, Zeyu

    One of the major challenges in the development of targeted nanoparticles (NPs) for cancer therapy is to discover targeting ligands that allow for differential binding and uptake by the target cancer cells. Using prostate ...

  17. Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics.

    PubMed

    Shalev, Moran; Rozenberg, Haim; Smolkin, Boris; Nasereddin, Abedelmajeed; Kopelyanskiy, Dmitry; Belakhov, Valery; Schrepfer, Thomas; Schacht, Jochen; Jaffe, Charles L; Adir, Noam; Baasov, Timor

    2015-09-30

    Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL. PMID:26264664

  18. Structural basis for selective targeting of leishmanial ribosomes: Aminoglycoside derivatives as promising therapeutics

    SciTech Connect

    Shalev, Moran; Rozenberg, Haim; Smolkin, Boris; Nasereddin, Abedelmajeed; Kopelyanskiy, Dmitry; Belakhov, Valery; Schrepfer, Thomas; Schacht, Jochen; Jaffe, Charles L.; Adir, Noam; Baasov, Timor

    2015-08-11

    Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)—the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3, as a prospective therapeutic candidate for the treatment of VL.

  19. Structural basis for selective targeting of leishmanial ribosomes: Aminoglycoside derivatives as promising therapeutics

    DOE PAGESBeta

    Shalev, Moran; Rozenberg, Haim; Smolkin, Boris; Nasereddin, Abedelmajeed; Kopelyanskiy, Dmitry; Belakhov, Valery; Schrepfer, Thomas; Schacht, Jochen; Jaffe, Charles L.; Adir, Noam; et al

    2015-08-11

    Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)—the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and hasmore »yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3, as a prospective therapeutic candidate for the treatment of VL.« less

  20. Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis

    PubMed Central

    Zhang, Hongquan; Wang, Zhaohui; Stupak, Jacek; Ghribi, Othman; Geiger, Jonathan D.; Liu, Qing Yan; Li, Jianjun

    2013-01-01

    The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study, we validated the method by analyzing globally N-glycans expressed in different systems; single proteins, cancer cells and serum samples. A dynamic range of three orders of magnitude was obtained for the detection of all five glycans released from ribonuclease B. The limit of detection of 80 attomole for Man9GlcNAc2 demonstrated the excellent sensitivity of the method. The capability of the strategy to identify diverse glycans was demonstrated by identification and detection of 162 different glycans and isomers from pancreatic cancer cells. The sensitivity of the method was illustrated further by the ability to detect 8 glycans from 250 cancer cells and 5 glycans released from 100 cancer cells. In serum obtained from rabbits fed control diet or diet enriched with 2% cholesterol, differences to 42 glycans were accurately measured and this indicates that this strategy might find use in studies of biomarker discovery and validation. PMID:22821818

  1. Characterisation of aptamer-target interactions by branched selection and high-throughput sequencing of SELEX pools.

    PubMed

    Dupont, Daniel M; Larsen, Niels; Jensen, Jan K; Andreasen, Peter A; Kjems, Jørgen

    2015-12-01

    Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10(16) different RNA or DNA sequences by 5-10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2'-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers. PMID:26163061

  2. Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

    PubMed Central

    Dupont, Daniel M.; Larsen, Niels; Jensen, Jan. K.; Andreasen, Peter A.; Kjems, Jørgen

    2015-01-01

    Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 1016 different RNA or DNA sequences by 5–10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2?-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers. PMID:26163061

  3. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  4. LAMP proteins are required for fusion of lysosomes with phagosomes.

    PubMed

    Huynh, Kassidy K; Eskelinen, Eeva-Liisa; Scott, Cameron C; Malevanets, Anatoly; Saftig, Paul; Grinstein, Sergio

    2007-01-24

    Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other. PMID:17245426

  5. 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

  6. 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

  7. Combined aerobic exercise and enzyme replacement therapy rejuvenates the mitochondrial-lysosomal axis and alleviates autophagic blockage in Pompe disease.

    PubMed

    Nilsson, M I; MacNeil, L G; Kitaoka, Y; Suri, R; Young, S P; Kaczor, J J; Nates, N J; Ansari, M U; Wong, T; Ahktar, M; Brandt, L; Hettinga, B P; Tarnopolsky, M A

    2015-10-01

    A unifying feature in the pathogenesis of aging, neurodegenerative disease, and lysosomal storage disorders is the progressive deposition of macromolecular debris impervious to enzyme catalysis by cellular waste disposal mechanisms (e.g., lipofuscin). Aerobic exercise training (AET) has pleiotropic effects and stimulates mitochondrial biogenesis, antioxidant defense systems, and autophagic flux in multiple organs and tissues. Our aim was to explore the therapeutic potential of AET as an ancillary therapy to mitigate autophagic buildup and oxidative damage and rejuvenate the mitochondrial-lysosomal axis in Pompe disease (GSD II/PD). Fourteen weeks of combined recombinant acid ?-glucosidase (rhGAA) and AET polytherapy attenuated mitochondrial swelling, fortified antioxidant defense systems, reduced oxidative damage, and augmented glycogen clearance and removal of autophagic debris/lipofuscin in fast-twitch skeletal muscle of GAA-KO mice. Ancillary AET potently augmented the pool of PI4KA transcripts and exerted a mild restorative effect on Syt VII and VAMP-5/myobrevin, collectively suggesting improved endosomal transport and Ca(2+)- mediated lysosomal exocytosis. Compared with traditional rhGAA monotherapy, AET and rhGAA polytherapy effectively mitigated buildup of protein carbonyls, autophagic debris/lipofuscin, and P62/SQSTM1, while enhancing MnSOD expression, nuclear translocation of Nrf-2, muscle mass, and motor function in GAA-KO mice. Combined AET and rhGAA therapy reactivates cellular clearance pathways, mitigates mitochondrial senescence, and strengthens antioxidant defense systems in GSD II/PD. Aerobic exercise training (or pharmacologic targeting of contractile-activity-induced pathways) may have therapeutic potential for mitochondrial-lysosomal axis rejuvenation in lysosomal storage disorders and related conditions (e.g., aging and neurodegenerative disease). PMID:26001726

  8. Melanoregulin (MREG) modulates lysosome function in pigment epithelial cells.

    PubMed

    Damek-Poprawa, Monika; Diemer, Tanja; Lopes, Vanda S; Lillo, Concepción; Harper, Dawn C; Marks, Michael S; Wu, Yalin; Sparrow, Janet R; Rachel, Rivka A; Williams, David S; Boesze-Battaglia, Kathleen

    2009-04-17

    Melanoregulin (MREG), the product of the Mreg(dsu) gene, is a small highly charged protein, hypothesized to play a role in organelle biogenesis due to its effect on pigmentation in dilute, ashen, and leaden mutant mice. Here we provide evidence that MREG is required in lysosome-dependent phagosome degradation. In the Mreg(-/-) mouse, we show that loss of MREG function results in phagosome accumulation due to delayed degradation of engulfed material. Over time, the Mreg(-/-) mouse retinal pigment epithelial cells accumulate the lipofuscin component, A2E. MREG-deficient human and mouse retinal pigment epithelial cells exhibit diminished activity of the lysosomal hydrolase, cathepsin D, due to defective processing. Moreover, MREG localizes to small intracellular vesicles and associates with the endosomal phosphoinositide, phosphatidylinositol 3,5-biphosphate. Collectively, these studies suggest that MREG is required for lysosome maturation and support a role for MREG in intracellular trafficking. PMID:19240024

  9. 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

  10. 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

  11. Lysosomal two-pore channel subtype 2 (TPC2) regulates skeletal muscle autophagic signaling.

    PubMed

    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

    2015-02-01

    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

  12. Targeting the binding interface on a shared receptor subunit of a cytokine family enables the inhibition of multiple member cytokines with selectable target spectrum.

    PubMed

    Nata, Toshie; Basheer, Asjad; Cocchi, Fiorenza; van Besien, Richard; Massoud, Raya; Jacobson, Steven; Azimi, Nazli; Tagaya, Yutaka

    2015-09-11

    The common ? molecule (?c) is a shared signaling receptor subunit used by six ?c-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple ?c-cytokines are pathogenically involved in a single disease, thus making the shared ?c-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the ?c and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the ?c and cytokines, we successfully designed peptides that not only inhibit multiple ?c-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three ?c-cytokines without affecting the other three or non-?c-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of ?c-cytokines the ?c-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of ?c-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases. PMID:26183780

  13. Epidemiology and diagnosis of lysosomal storage disorders; challenges of screening.

    PubMed

    Kingma, Sandra D K; Bodamer, Olaf A; Wijburg, Frits A

    2015-03-01

    The lysosomal storage disorders (LSDs) are a group of genetic disorders resulting from defective lysosomal metabolism and subsequent accumulation of substrates. Patients present with a large phenotypic spectrum of disease manifestations that are generally not specific for LSDs, leading to considerable diagnostic delay and missed cases. Introduction of new disease modifying therapies for LSDs has made early diagnosis a priority. Increased awareness, but particularly the introduction of screening programs allow for early diagnosis and timely initiation of treatment. This review will provide insight into the epidemiology and diagnostic process for LSDs. In addition, challenges for carrier screening, high-risk screening and newborn population screening for LSDs are discussed. PMID:25987169

  14. Lysosome-related organelles: Unusual compartments become mainstream

    PubMed Central

    Marks, Michael S.; Heijnen, Harry F. G.; Raposo, Graça

    2013-01-01

    Lysosome-related organelles (LROs) comprise a group of cell type-specific subcellular compartments with unique composition, morphology and structure that share some features with endosomes and lysosomes and that function in varied processes such as pigmentation, hemostasis, lung plasticity and immunity. In recent years, studies of genetic diseases in which LRO functions are compromised have provided new insights into the mechanisms of LRO biogenesis and the regulated secretion of LRO contents. These insights have revealed previously unappreciated specialized endosomal sorting processes in all cell types, and are expanding our views of the plasticity of the endosomal and secretory systems in adapting to cell type-specific needs. PMID:23726022

  15. Selective induction of oxidative stress in cancer cells via synergistic combinations of agents targeting redox homeostasis.

    PubMed

    Akladios, Fady N; Andrew, Scott D; Parkinson, Christopher J

    2015-07-01

    Cancer cell resistance to chemotherapy is still a heavy burden that impairs the response of many cancer patients to conventional chemotherapy. Using drug combinations is one therapeutic approach to overcome the developing resistance to any one drug. Oxidative stress is now a generally regarded hallmark of cancer that can be one approach to selectively target cancer cells while sparing normal cells. With the aim of increasing oxidative stress in cancer cells to a lethal set point, we have generated and combined several series of redox active compounds that act at different points of the cellular oxidative cascade. The premise of such combinations is to deplete of endogenous antioxidant defence proteins (e.g., Glutathione) while concomitantly increasing the generation of ROS via metal redox recycling and Fenton chemistry which eventually leads to the disruption of cellular redox homeostasis and induction of cell death. Through this approach, we have identified highly synergistic combinations of two distinctive classes of compounds (Azines and Copper(II) complexes of 2-pyridyl ketone thiosemicarbazones) which are capable of eliminating cancer cells without concomitant increase in toxicity toward normal cells. In one of our most potent combinations, a combination index (CI) value of 0.056 was observed, representing a 17 fold enhancement in activity beyond additive effects. Such new combination regimen of redox active compounds can be one step closer to potentially safer low dose chemotherapy. PMID:26022081

  16. 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

  17. An Assessment of the Effect of Rotenone on Selected Non-Target Aquatic Fauna

    PubMed Central

    Dalu, Tatenda; Wasserman, Ryan J.; Jordaan, Martine; Froneman, William P.; Weyl, Olaf L. F.

    2015-01-01

    Rotenone, a naturally occurring ketone, is widely employed for the management of invasive fish species. The use of rotenone poses serious challenges to conservation practitioners due to its impacts on non-target organisms including amphibians and macroinvertebrates. Using laboratory studies, we investigated the effects of different rotenone concentrations (0, 12.5, 25, 37.5, 50, 100 ?g L-1) on selected invertebrate groups; Aeshnidae, Belostomatids, Decapods, Ephemeroptera, Pulmonata and zooplankton over a period of 18 hours. Based on field observations and body size, we hypothesized that Ephemeropterans and zooplankton would be more susceptible to rotenone than Decapods, Belostomatids and snails. Experimental results supported this hypothesis and mortality and behaviour effects varied considerably between taxa, ranging from no effect (crab Potamonuates sidneyi) to 100% mortality (Daphnia pulex and Paradiaptomus lamellatus). Planktonic invertebrates were particularly sensitive to rotenone even at very low concentrations. Future research should investigate the recovery time of invertebrate communities after the application of rotenone and conduct field assessments assessing the longer term effects of rotenone exposure on the population dynamics of those less sensitive organisms. PMID:26540301

  18. Selective BCL-2 Inhibition by ABT-199 Causes On Target Cell Death in Acute Myeloid Leukemia

    PubMed Central

    Pan, Rongqing; Hogdal, Leah J.; Benito, Juliana M; Bucci, Donna; Han, Lina; Borthakur, Gautam; Cortes, Jorge; DeAngelo, Daniel J.; Debose, LaKeisha; Mu, Hong; Döhner, Hartmut; Gaidzik, Verena I.; Galinsky, Ilene; Golfman, Leonard S.; Haferlach, Torsten; Harutyunyan, Karine G.; Hu, Jianhua; Leverson, Joel D; Marcucci, Guido; Müschen, Markus; Newman, Rachel; Park, Eugene; Ruvolo, Peter P.; Ruvolo, Vivian; Ryan, Jeremy; Schindela, Sonja; Zweidler-McKay, Patrick; Stone, Richard M.; Kantarjian, Hagop; Andreeff, Michael; Konopleva, Marina; Letai, Anthony G.

    2014-01-01

    B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nM, and cell death occurred within 2 h. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia (CLL), a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML. PMID:24346116

  19. Selectively targeting an inactive conformation of interleukin-2-inducible T-cell kinase by allosteric inhibitors.

    PubMed

    Han, Seungil; Czerwinski, Robert M; Caspers, Nicole L; Limburg, David C; Ding, WeiDong; Wang, Hong; Ohren, Jeffrey F; Rajamohan, Francis; McLellan, Thomas J; Unwalla, Ray; Choi, Chulho; Parikh, Mihir D; Seth, Nilufer; Edmonds, Jason; Phillips, Chris; Shakya, Subarna; Li, Xin; Spaulding, Vikki; Hughes, Samantha; Cook, Andrew; Robinson, Colin; Mathias, John P; Navratilova, Iva; Medley, Quintus G; Anderson, David R; Kurumbail, Ravi G; Aulabaugh, Ann

    2014-06-01

    ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays. PMID:24593284

  20. Selective Capture and Quick Detection of Targeting Cells with SERS-Coding Microsphere Suspension Chip.

    PubMed

    Li, Dian; Zhang, Yuting; Li, Ruimin; Guo, Jia; Wang, Changchun; Tang, Chuanbing

    2015-05-13

    Circulating tumor cells (CTCs) captured from blood fluid represent recurrent cancers and metastatic lesions to monitor the situation of cancers. We develop surface-enhanced Raman scattering (SERS)-coding microsphere suspension chip as a new strategy for fast and efficient capture, recovery, and detection of targeting cancer cells. Using HeLa cells as model CTCs, we first utilize folate as a recognition molecule to be immobilized in magnetic composite microspheres for capturing HeLa cells and attaining high capturing efficacy (up to 95%). After capturing cells, the composite microsphere, which utilizes a disulfide bond as crosslinker in the polymer shell and as a spacer for linking folate, can recycle 90% cells within 20 min eluted by glutathion solution. Taking advantage of the SERS with fingerprint features, we characterize captured/recovered cells with the unique signal of report-molecule 4-aminothiophenol through introducing the SERS-coding microsphere suspension chip to CTCs. Finally, the exploratory experiment of sieving cells shows that the magnetic composite microspheres can selectively capture the HeLa cells from samples of mixed cells, indicating that these magnetic composite microspheres have potential in real blood samples for capturing CTCs. PMID:25597293

  1. An Assessment of the Effect of Rotenone on Selected Non-Target Aquatic Fauna.

    PubMed

    Dalu, Tatenda; Wasserman, Ryan J; Jordaan, Martine; Froneman, William P; Weyl, Olaf L F

    2015-01-01

    Rotenone, a naturally occurring ketone, is widely employed for the management of invasive fish species. The use of rotenone poses serious challenges to conservation practitioners due to its impacts on non-target organisms including amphibians and macroinvertebrates. Using laboratory studies, we investigated the effects of different rotenone concentrations (0, 12.5, 25, 37.5, 50, 100 ?g L-1) on selected invertebrate groups; Aeshnidae, Belostomatids, Decapods, Ephemeroptera, Pulmonata and zooplankton over a period of 18 hours. Based on field observations and body size, we hypothesized that Ephemeropterans and zooplankton would be more susceptible to rotenone than Decapods, Belostomatids and snails. Experimental results supported this hypothesis and mortality and behaviour effects varied considerably between taxa, ranging from no effect (crab Potamonuates sidneyi) to 100% mortality (Daphnia pulex and Paradiaptomus lamellatus). Planktonic invertebrates were particularly sensitive to rotenone even at very low concentrations. Future research should investigate the recovery time of invertebrate communities after the application of rotenone and conduct field assessments assessing the longer term effects of rotenone exposure on the population dynamics of those less sensitive organisms. PMID:26540301

  2. Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation

    PubMed Central

    Lee, Yin-Fai; Tawfik, Dan S.; Griffiths, Andrew D.

    2002-01-01

    In vitro compartmentalisation (IVC), a technique for selecting genes encoding enzymes based on compartmentalising gene translation and enzymatic reactions in emulsions, was used to investigate the interaction of the DNA cytosine-5 methyltransferase M.HhaI with its target DNA (5?-GCGC-3?). Crystallog raphy shows that the active site loop from the large domain of M.HhaI interacts with a flipped-out cytosine (the target for methylation) and two target recognition loops (loops I and II) from the small domain make almost all the other base-specific interactions. A library of M.HhaI genes was created by randomising all the loop II residues thought to make base-specific interactions and directly determine target specificity. The library was selected for 5?-GCGC-3? methylation. Interestingly, in 11 selected active clones, 10 different sequences were found and none were wild-type. At two of the positions mutated (Ser252 and Tyr254) a number of different amino acids could be tolerated. At the third position, however, all active mutants had a glycine, as in wild-type M.HhaI, suggesting that Gly257 is crucial for DNA recognition and enzyme activity. Our results suggest that recognition of base pairs 3 and 4 of the target site either relies entirely on main chain interactions or that different residues from those identified in the crystal structure contribute to DNA recognition. PMID:12433997

  3. Speed-selectivity paradox in the protein search for targets on DNA: is it real or not?

    PubMed

    Veksler, Alex; Kolomeisky, Anatoly B

    2013-10-24

    Protein search for targets on DNA starts all major biological processes. Although significant experimental and theoretical efforts have been devoted to investigation of these phenomena, mechanisms of protein-DNA interactions during the search remain not fully understood. One of the most surprising observations is known as a speed-selectivity paradox. It suggests that experimentally observed fast findings of targets require smooth protein-DNA binding potentials, while the stability of the specific protein-DNA complex imposes a large energy gap which should significantly slow down the protein molecule. We developed a discrete-state stochastic approach that allowed us to investigate explicitly target search phenomena and to analyze the speed-selectivity paradox. A general dynamic phase diagram for different search regimes is constructed. The effect of the target position on search dynamics is investigated. Using experimentally observed parameters, it is found that slow protein diffusion on DNA does not lead to an increase in the search times. Thus, our theory resolves the speed-selectivity paradox by arguing that it does not exist. It is just an artifact of using approximate continuum theoretical models for analyzing protein search in the region of the parameter space beyond the range of validity of these models. In addition, the presented method, for the first time, provides an explanation for fast target search at the level of single protein molecules. Our theoretical predictions agree with all available experimental observations, and extensive Monte Carlo computer simulations are performed to support analytical calculations. PMID:23316873

  4. Precipitation effects on the selection of suitable non-variant targets intended for atmospheric correction of satellite remotely sensed imagery

    NASA Astrophysics Data System (ADS)

    Themistocleous, Kyriacos; Hadjimitsis, Diofantos G.; Retalis, Adrianos; Chrysoulakis, Nektarios; Michaelides, Silas

    2013-09-01

    One of the most well-established atmospheric correction methods of satellite imagery is the use of the empirical line method using non-variant targets. Non-variant targets serve as pseudo-invariant targets since their reflectance values are stable across time. A recent adaptation of the empirical line method incorporates the use of ground reflectance measurements of selected non-variant targets. Most of the users are not aware of the existing conditions of the pseudo-invariant targets; i.e., whether they are dry or wet. Any omission of such effects may cause erroneous results; therefore, remote sensing users must be aware of such effects. This study assessed the effects of precipitation on five types of commonly located surfaces, including asphalt, concrete and sand, intended as pseudo-invariant targets for atmospheric correction. Spectroradiometric measurements were taken in wet and dry conditions to obtain the spectral signatures of the targets, from January 2010 to May 2011 (46 campaigns). An atmospheric correction of eleven Landsat TM/ETM + satellite images using the empirical line method was conducted. To identify the effects of precipitation, a comparison was conducted of the atmospheric path radiance component for wet and dry conditions. It was found that precipitation conditions such as rainfall affected the reflectance values of the surfaces, especially sand. Therefore, precipitation conditions need to be considered when using non-variant targets in atmospheric correction methods.

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

    E-print Network

    Abidi, Mongi A.

    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

  6. TouchCuts and TouchZoom: Enhanced Target Selection for Touch Displays using Finger Proximity Sensing

    E-print Network

    touch-screen laptops are increasing in popularity, users still do not comfortably rely on touch have started producing laptops equipped with touch-screens, allowing users to use their fingersTouchCuts and TouchZoom: Enhanced Target Selection for Touch Displays using Finger Proximity

  7. 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,…

  8. Lysosomal cobalamin accumulation in fibroblasts from a patient with an inborn error of cobalamin metabolism (cblF complementation group): Visualization by electron microscope radioautography

    SciTech Connect

    Vassiliadis, A.; Rosenblatt, D.S.; Cooper, B.A.; Bergeron, J.J.M. )

    1991-08-01

    Cobalamin (Cbl, vitamin B{sub 12}) bound to transcobalamin II (TCII) enters cultured fibroblasts by receptor-mediated endocytosis. Following degradation of the TCII, Cbl is subsequently found in either the cytoplasm bound to methionine synthase or in the mitochondria bound to methylmalonyl CoA mutase. In fibroblasts from patients belonging to the cblF complementation group, Cbl is found free in the cell and is not transferred to the above two target enzymes. Quantitative Em radioautography was utilized to visualize intracellular Cbl in fibroblasts from cblF patients and from normal subjects. In cblF cells, 60% of all silver grains were assigned to lysosomes, with only 12.6% over cytoplasm and 1.2% over mitochondria. Subcellular fractionation showed that in cblF cells, the majority of label was associated with clearly recognizable lysosomes. These studies conclusively demonstrate that secondary lysosomes accumulate Cbl in cblF disease.

  9. Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1.

    PubMed

    Zhang, Zhongsheng; Ojo, Kayode K; Johnson, Steven M; Larson, Eric T; He, Penqing; Geiger, Jennifer A; Castellanos-Gonzalez, Alejandro; White, A Clinton; Parsons, Marilyn; Merritt, Ethan A; Maly, Dustin J; Verlinde, Christophe L M J; Van Voorhis, Wesley C; Fan, Erkang

    2012-08-15

    Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues. PMID:22795629

  10. Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1

    PubMed Central

    Zhang, Zhongsheng; Ojo, Kayode K.; Johnson, Steven M.; Larson, Eric T.; He, Penqing; Geiger, Jennifer A.; Castellanos-Gonzalez, Alejandro; White, A. Clinton; Parsons, Marilyn; Merritt, Ethan A.; Maly, Dustin J.; Verlinde, Christophe L. M. J.; Van Voorhis, Wesley C.; Fan, Erkang

    2012-01-01

    Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues. PMID:22795629

  11. Genomic expression analyses reveal lysosomal, innate immunity proteins, as disease correlates in murine models of a lysosomal storage disorder.

    PubMed

    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 Npc1(nmf164) 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. 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…

  13. Attention Blinks for Selection, Not Perception or Memory: Reading Sentences and Reporting Targets

    ERIC Educational Resources Information Center

    Potter, Mary C.; Wyble, Brad; Olejarczyk, Jennifer

    2011-01-01

    In whole report, a sentence presented sequentially at the rate of about 10 words/s can be recalled accurately, whereas if the task is to report only two target words (e.g., red words), the second target suffers an attentional blink if it appears shortly after the first target. If these two tasks are carried out simultaneously, is there an…

  14. SLC17A9 Protein Functions as a Lysosomal ATP Transporter and Regulates Cell Viability*

    PubMed Central

    Cao, Qi; Zhao, Kexin; Zhong, Xi Zoë; Zou, Yuanjie; Yu, Haichuan; Huang, Peng; Xu, Tian-Le; Dong, Xian-Ping

    2014-01-01

    Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability. PMID:24962569

  15. APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability

    PubMed Central

    Lan, Xiqian; Jhaveri, Aakash; Cheng, Kang; Wen, Hongxiu; Saleem, Moin A.; Mathieson, Peter W.; Mikulak, Joanna; Aviram, Sharon; Malhotra, Ashwani; Skorecki, Karl

    2014-01-01

    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

  16. Photoactivation of lysosomally sequestered sunitinib after angiostatic treatment causes vascular occlusion and enhances tumor growth inhibition

    PubMed Central

    Nowak-Sliwinska, P; Weiss, A; van Beijnum, J R; Wong, T J; Kilarski, W W; Szewczyk, G; Verheul, H M W; Sarna, T; van den Bergh, H; Griffioen, A W

    2015-01-01

    The angiogenesis inhibitor sunitinib is a tyrosine kinase inhibitor that acts mainly on the VEGF and PDGF pathways. We have previously shown that sunitinib is sequestered in the lysosomes of exposed tumor and endothelial cells. This phenomenon is part of the drug-induced resistance observed in the clinic. Here, we demonstrate that when exposed to light, sequestered sunitinib causes immediate destruction of the lysosomes, resulting in the release of sunitinib and cell death. We hypothesized that this photoactivation of sunitinib could be used as a vaso-occlusive vascular-targeting approach to treating cancer. Spectral properties of sunitinib and its lysosomal accumulation were measured in vitro. The human A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane (CAM) and the Colo-26 colorectal carcinoma model in Balb/c mice were used to test the effects of administrating sunitinib and subsequently exposing tumor tissue to light. Tumors were subsequently resected and subject to immunohistochemical analysis. In A2780 ovarian carcinoma tumors, treatment with sunitinib+light resulted in immediate specific angio-occlusion, leading to a necrotic tumor mass 24?h after treatment. Tumor growth was inhibited by 70% as compared with the control group (**P<0.0001). Similar observations were made in the Colo-26 colorectal carcinoma, where light exposure of the sunitinib-treated mice inhibited tumor growth by 50% as compared with the control and by 25% as compared with sunitinib-only-treated tumors (N?4; P=0.0002). Histology revealed that photoactivation of sunitinib resulted in a change in tumor vessel architecture. The current results suggest that the spectral properties of sunitinib can be exploited for application against certain cancer indications. PMID:25675301

  17. Developmental Cell A TRP Channel in the Lysosome Regulates

    E-print Network

    Xu, Haoxing

    Developmental Cell Article A TRP Channel in the Lysosome Regulates Large Particle Phagocytosis via Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular blood cells (RBCs), through a highly regu- lated process called phagocytosis (Flannagan et al., 2012

  18. Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy.

    PubMed

    Baulies, Anna; Ribas, Vicent; Núñez, Susana; Torres, Sandra; Alarcón-Vila, Cristina; Martínez, Laura; Suda, Jo; Ybanez, Maria D; Kaplowitz, Neil; García-Ruiz, Carmen; Fernández-Checa, Jose C

    2015-01-01

    The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)(-/-) mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase(+/+) littermates. ASMase(-/-) hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase(+/+) hepatocytes caused by U18666A reproduces the susceptibility of ASMase(-/-) hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase(-/-) mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol ?-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury. PMID:26657973

  19. A model of lysosomal pH regulation

    PubMed Central

    Ishida, Yoichi; Nayak, Smita

    2013-01-01

    Lysosomes must maintain an acidic luminal pH to activate hydrolytic enzymes and degrade internalized macromolecules. Acidification requires the vacuolar-type H+-ATPase to pump protons into the lumen and a counterion flux to neutralize the membrane potential created by proton accumulation. Early experiments suggested that the counterion was chloride, and more recently a pathway consistent with the ClC-7 Cl–/H+ antiporter was identified. However, reports that the steady-state luminal pH is unaffected in ClC-7 knockout mice raise questions regarding the identity of the carrier and the counterion. Here, we measure the current–voltage characteristics of a mammalian ClC-7 antiporter, and we use its transport properties, together with other key ion regulating elements, to construct a mathematical model of lysosomal pH regulation. We show that results of in vitro lysosome experiments can only be explained by the presence of ClC-7, and that ClC-7 promotes greater acidification than Cl–, K+, or Na+ channels. Our models predict strikingly different lysosomal K+ dynamics depending on the major counterion pathways. However, given the lack of experimental data concerning acidification in vivo, the model cannot definitively rule out any given mechanism, but the model does provide concrete predictions for additional experiments that would clarify the identity of the counterion and its carrier. PMID:23712550

  20. 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,…

  1. 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

  2. Structure of human saposin A at lysosomal pH

    SciTech Connect

    Hill, Chris H.; Read, Randy J.; Deane, Janet E.

    2015-06-27

    A 1.8 Å resolution structure of the sphingolipid activator protein saposin A has been determined at pH 4.8, the physiologically relevant lysosomal pH for hydrolase enzyme activation and lipid-transfer activity. The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme ?-galactocerebrosidase (GALC), which catalyzes the breakdown of ?-d-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a ‘closed’ to an ‘open’ conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8 Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined ‘closed’ conformation, showing that pH alone is not sufficient for the transition to the ‘open’ conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility.

  3. Lysosomal ?-glucuronidase regulates Lyme and rheumatoid arthritis severity

    PubMed Central

    Bramwell, Kenneth K.C.; Ma, Ying; Weis, John H.; Chen, Xinjian; Zachary, James F.; Teuscher, Cory; Weis, Janis J.

    2013-01-01

    Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent arthropod-borne illness in the United States and remains a clinical and social challenge. The spectrum of disease severity among infected patients suggests that host genetics contribute to pathogenic outcomes, particularly in patients who develop arthritis. Using a forward genetics approach, we identified the lysosomal enzyme ?-glucuronidase (GUSB), a member of a large family of coregulated lysosomal enzymes, as a key regulator of Lyme-associated arthritis severity. Severely arthritic C3H mice possessed a naturally occurring hypomorphic allele, Gusbh. C57BL/6 mice congenic for the C3H Gusb allele were prone to increased Lyme-associated arthritis severity. Radiation chimera experiments revealed that resident joint cells drive arthritis susceptibility. C3H mice expressing WT Gusb as a transgene were protected from severe Lyme arthritis. Importantly, the Gusbh allele also exacerbated disease in a serum transfer model of rheumatoid arthritis. A known GUSB function is the prevention of lysosomal accumulation of glycosaminoglycans (GAGs). Development of Lyme and rheumatoid arthritis in Gusbh-expressing mice was associated with heightened accumulation of GAGs in joint tissue. We propose that GUSB modulates arthritis pathogenesis by preventing accumulation of proinflammatory GAGs within inflamed joint tissue, a trait that may be shared by other lysosomal exoglycosidases. PMID:24334460

  4. Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy

    PubMed Central

    Baulies, Anna; Ribas, Vicent; Núñez, Susana; Torres, Sandra; Alarcón-Vila, Cristina; Martínez, Laura; Suda, Jo; Ybanez, Maria D.; Kaplowitz, Neil; García-Ruiz, Carmen; Fernández-Checa, Jose C.

    2015-01-01

    The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)?/? mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase+/+ littermates. ASMase?/? hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase+/+ hepatocytes caused by U18666A reproduces the susceptibility of ASMase?/? hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase?/? mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol ?-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury. PMID:26657973

  5. 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.

  6. 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.; Gonzalez-Hernandez, I.

    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.

  7. NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses

    PubMed Central

    Cheng, Yuan; Chi, Ying-Jun; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2013-01-01

    Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions. PMID:23341779

  8. SynLethDB: synthetic lethality database toward discovery of selective and sensitive anticancer drug targets.

    PubMed

    Guo, Jing; Liu, Hui; Zheng, Jie

    2016-01-01

    Synthetic lethality (SL) is a type of genetic interaction between two genes such that simultaneous perturbations of the two genes result in cell death or a dramatic decrease of cell viability, while a perturbation of either gene alone is not lethal. SL reflects the biologically endogenous difference between cancer cells and normal cells, and thus the inhibition of SL partners of genes with cancer-specific mutations could selectively kill cancer cells but spare normal cells. Therefore, SL is emerging as a promising anticancer strategy that could potentially overcome the drawbacks of traditional chemotherapies by reducing severe side effects. Researchers have developed experimental technologies and computational prediction methods to identify SL gene pairs on human and a few model species. However, there has not been a comprehensive database dedicated to collecting SL pairs and related knowledge. In this paper, we propose a comprehensive database, SynLethDB (http://histone.sce.ntu.edu.sg/SynLethDB/), which contains SL pairs collected from biochemical assays, other related databases, computational predictions and text mining results on human and four model species, i.e. mouse, fruit fly, worm and yeast. For each SL pair, a confidence score was calculated by integrating individual scores derived from different evidence sources. We also developed a statistical analysis module to estimate the druggability and sensitivity of cancer cells upon drug treatments targeting human SL partners, based on large-scale genomic data, gene expression profiles and drug sensitivity profiles on more than 1000 cancer cell lines. To help users access and mine the wealth of the data, we developed other practical functionalities, such as search and filtering, orthology search, gene set enrichment analysis. Furthermore, a user-friendly web interface has been implemented to facilitate data analysis and interpretation. With the integrated data sets and analytics functionalities, SynLethDB would be a useful resource for biomedical research community and pharmaceutical industry. PMID:26516187

  9. SynLethDB: synthetic lethality database toward discovery of selective and sensitive anticancer drug targets

    PubMed Central

    Guo, Jing; Liu, Hui; Zheng, Jie

    2016-01-01

    Synthetic lethality (SL) is a type of genetic interaction between two genes such that simultaneous perturbations of the two genes result in cell death or a dramatic decrease of cell viability, while a perturbation of either gene alone is not lethal. SL reflects the biologically endogenous difference between cancer cells and normal cells, and thus the inhibition of SL partners of genes with cancer-specific mutations could selectively kill cancer cells but spare normal cells. Therefore, SL is emerging as a promising anticancer strategy that could potentially overcome the drawbacks of traditional chemotherapies by reducing severe side effects. Researchers have developed experimental technologies and computational prediction methods to identify SL gene pairs on human and a few model species. However, there has not been a comprehensive database dedicated to collecting SL pairs and related knowledge. In this paper, we propose a comprehensive database, SynLethDB (http://histone.sce.ntu.edu.sg/SynLethDB/), which contains SL pairs collected from biochemical assays, other related databases, computational predictions and text mining results on human and four model species, i.e. mouse, fruit fly, worm and yeast. For each SL pair, a confidence score was calculated by integrating individual scores derived from different evidence sources. We also developed a statistical analysis module to estimate the druggability and sensitivity of cancer cells upon drug treatments targeting human SL partners, based on large-scale genomic data, gene expression profiles and drug sensitivity profiles on more than 1000 cancer cell lines. To help users access and mine the wealth of the data, we developed other practical functionalities, such as search and filtering, orthology search, gene set enrichment analysis. Furthermore, a user-friendly web interface has been implemented to facilitate data analysis and interpretation. With the integrated data sets and analytics functionalities, SynLethDB would be a useful resource for biomedical research community and pharmaceutical industry. PMID:26516187

  10. Motexafin gadolinium, a tumor-selective drug targeting thioredoxin reductase and ribonucleotide reductase.

    PubMed

    Hashemy, Seyed Isaac; Ungerstedt, Johanna S; Zahedi Avval, Farnaz; Holmgren, Arne

    2006-04-21

    Motexafin gadolinium (MGd) is a chemotherapeutic drug that selectively targets tumor cells and mediates redox reactions generating reactive oxygen species. Thioredoxin (Trx), NADPH, and thioredoxin reductase (TrxR) of the cytosol/nucleus or mitochondria are major thiol-dependent reductases with many functions in cell growth, defense against oxidative stress, and apoptosis. Mammalian TrxRs are selenocysteine-containing flavoenzymes; MGd was an NADPH-oxidizing substrate for human or rat TrxR1 with a Km value of 8.65 microM (kcat/Km of 4.86 x 10(4) M(-1) s(-1)). The reaction involved redox cycling of MGd by oxygen producing superoxide and hydrogen peroxide. MGd acted as a non-competitive inhibitor (IC50 of 6 microM) for rat TrxR. In contrast, direct reaction between MGd and reduced human Trx was negligible. The corresponding reaction with reduced Escherichia coli Trx was also negligible, but MGd was a better substrate (kcat/Km of 2.23 x 10(5) M(-1) s(-1)) for TrxR from E. coli and a strong inhibitor of Trx-dependent protein disulfide reduction. Ribonucleotide reductase (RNR), a 1:1 complex of the non-identical R1- and R2-subunits, catalyzes the essential de novo synthesis of deoxyribonucleotides for DNA synthesis using electrons from Trx and TrxR. MGd inhibited recombinant mouse RNR activity with either 3 microM reduced human Trx (IC50 2 microM) or 4 mM dithiothreitol (IC50 6 microM) as electron donors. Our results demonstrate MGd-induced enzymatic generation of reactive oxygen species by TrxR plus a powerful inhibition of RNR. This may explain the effects of the drug on cancer cells, which often overproduce TrxR and have induced RNR for replication and repair. PMID:16481328

  11. Lysosome size, motility and stress response regulated by Fronto-Temporal Dementia modifier TMEM106B

    PubMed Central

    Stagi, Massimiliano; Klein, Zoe A.; Gould, Travis J.; Bewersdorf, Joerg; Strittmatter, Stephen M.

    2014-01-01

    Fronto-Temporal Lobar Degeneration with TDP-43 (FTLD-TDP) is a fatal neurodegeneration. TMEM106B variants are linked to FTLD-TDP risk, and TMEM106B is lysosomal. Here, we focus on neuronal TMEM106B, and demonstrate co-localization and traffic with lysosomal LAMP-1. pH-sensitive reporters demonstrate that the TMEM106B C-terminus is lumenal. The TMEM106B N-terminus interacts with endosomal adaptors and other TMEM106 proteins. TMEM106B knockdown reduces neuronal lysosomal number and diameter by STED microscopy, and overexpression enlarges LAMP-positive structures. Reduction of TMEM106B increases axonally transported lysosomes, while TMEM106B elevation inhibits transport and yields large lysosomes in the soma. TMEM106B overexpression alters lysosomal stress signaling, causing a translocation of the mTOR-sensitive transcription factor, TFEB, to neuronal nuclei. TMEM106B loss-of-function delays TFEB translocation after Torin-1-induced stress. Enlarged TMEM106B-overexpressing lysosomes maintain organelle integrity longer after lysosomal photodamage than do control lysosomes, while small TMEM106B-knockdown lysosomes are more sensitive to illumination. Thus, neuronal TMEM106B plays a central role in regulating lysosomal size, motility and responsiveness to stress, highlighting the possible role of lysosomal biology in FTLD-TDP. PMID:25066864

  12. The influence of oxidation of membrane thiol groups on lysosomal proton permeability.

    PubMed Central

    Wan, F Y; Wang, Y N; Zhang, G J

    2001-01-01

    The influence of oxidation of membrane thiol groups on lysosomal proton permeability was studied by measuring lysosomal pH with FITC-conjugated dextran, determining the membrane potential with 3,3'-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Residual membrane thiol groups were measured with 5,5'-dithiobis-(2-nitrobenzoic acid). The lysosomal membrane thiol groups were modified by treatment with diamide and dithiothreitol. SDS/PAGE revealed aggregations of the membrane proteins induced by the treatment of lysosomes with diamide. The cross-linkage of proteins could be abolished by subsequent treatment with dithiothreitol, indicating that the proteins were linked via disulphide bonds. Treating the lysosomes with diamide decreased their membrane thiol groups and caused increases in lysosomal pH, membrane potential and proton leakage, which could be reversed by treatment of the lysosomes with dithiothreitol. This indicates that the lysosomal proton permeability can be increased by oxidation of the membrane thiol groups and restored to the normal level by reduction of the groups. Treatment of the lysosomes with N-ethylmaleimide reduced their membrane thiol groups but did not change the lysosomal pH or their degree of proton leakage. It suggests that protein aggregation may be an important mechanism for the increase in lysosomal proton permeability. The results raise the possibility that the proton permeability of lysosomes in vivo may be affected by the redox states of their membrane thiol groups. PMID:11716763

  13. The role of the frontal eye fields in oculomotor competition: image-guided TMS enhances contralateral target selection.

    PubMed

    Bosch, S E; Neggers, S F W; Van der Stigchel, S

    2013-04-01

    In order to execute a correct eye movement to a target in a search display, a saccade program toward the target element must be activated, while saccade programs toward distracting elements must be inhibited. The aim of the present study was to elucidate the role of the frontal eye fields (FEFs) in oculomotor competition. Functional magnetic resonance imaging-guided single-pulse transcranial magnetic stimulation (TMS) was administered over either the left FEF, the right FEF, or the vertex (control site) at 3 time intervals after target presentation, while subjects performed an oculomotor capture task. When TMS was applied over the FEF contralateral to the visual field where a target was presented, there was less interference of an ipsilateral distractor compared with FEF stimulation ipsilateral to the target's visual field or TMS over vertex. Furthermore, TMS over the FEFs decreased latencies of saccades to the contralateral visual field, irrespective of whether the saccade was directed to the target or to the distractor. These findings show that single-pulse TMS over the FEFs enhances the selection of a target in the contralateral visual field and decreases saccade latencies to the contralateral visual field. PMID:22455840

  14. Ets1 identified as a novel molecular target of RNA aptamer selected against metastatic cells for targeted delivery of nano-formulation

    PubMed Central

    Kaur, J; Tikoo, K

    2015-01-01

    Nanomedicine era is not far from its realization, but a major concern of targeted delivery still stands tall in its way. Herein we demonstrate the mechanism underlying the anticancer activity of an RNA aptamer (Apt) conjugated to gefitinib-loaded poly (lactic co-glycolic acid) nanoparticles (GNPs). Apt was selected through Cell-SELEX (systemic evolution of ligands by exponential enrichment) process against gefitinib-resistant H1975 lung cancer cells. The selected aptamer exhibited high specificity toward H1975 cells, both qualitatively as well as quantitatively. Software analysis using the MATCH tool predicted Ets1, a proto-oncoprotein, to be the target of the selected aptamer. Interestingly, the localization of identified aptamer varied in descending order of Ets1 expression, wherein maximum localization was observed in H1975 cells than in MDA-MB231, DU-145, H23, H460, A431, A549 and MCF-7 cells, and minimum in L132 cells. Furthermore, Apt-GNP bio-conjugate showed augmented anticancer activity specifically in Ets1-overexpressing cells. In addition, partial depletion of Ets1 in H1975 cells and overexpression of Ets1 in L132 cells reversed the targeting efficacy of the aptamer. Notably, a single intratumoral injection of the Apt-GNP bio-conjugate abrogated the growth of tumor in H1975 xenograft nude mice. Altogether, we present a pioneering platform, involving aptamers, which can be clinically used as a diagnostic marker for metastasis as well as an effective delivery system to escort the pharmaceutical cargo specifically to Ets1-overexpressing highly progressive tumors. PMID:25639877

  15. 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.

    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.

  16. Comparing the selection and placement of best management practices in improving water quality using a multiobjective optimization and targeting method.

    PubMed

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

    2014-03-01

    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

  17. Identification and Characterization of Genes Involved in Leishmania Pathogenesis: The Potential for Drug Target Selection

    PubMed Central

    Duncan, Robert; Gannavaram, Sreenivas; Dey, Ranadhir; Debrabant, Alain; Lakhal-Naouar, Ines; Nakhasi, Hira L.

    2011-01-01

    Identifying and characterizing Leishmania donovani genes and the proteins they encode for their role in pathogenesis can reveal the value of this approach for finding new drug targets. Effective drug targets are likely to be proteins differentially expressed or required in the amastigote life cycle stage found in the patient. Several examples and their potential for chemotherapeutic disruption are presented. A pathway nearly ubiquitous in living cells targeted by anticancer drugs, the ubiquitin system, is examined. New findings in ubiquitin and ubiquitin-like modifiers in Leishmania show how disruption of those pathways could point to additional drug targets. The programmed cell death pathway, now recognized among protozoan parasites, is reviewed for some of its components and evidence that suggests they could be targeted for antiparasitic drug therapy. Finally, the endoplasmic reticulum quality control system is involved in secretion of many virulence factors. How disruptions in this pathway reduce virulence as evidence for potential drug targets is presented. PMID:22091403

  18. Lysosome-controlled efficient ROS overproduction against cancer cells with a high pH-responsive catalytic nanosystem

    NASA Astrophysics Data System (ADS)

    Fu, Jingke; Shao, Yiran; Wang, Liyao; Zhu, Yingchun

    2015-04-01

    Excess reactive oxygen species (ROS) have been proved to damage cancer cells efficiently. ROS overproduction is thus greatly desirable for cancer therapy. To date, ROS production is generally uncontrollable and outside cells, which always bring severe side-effects in the vasculature. Since most ROS share a very short half-life and primarily react close to their site of formation, it would be more efficient if excess ROS are controllably produced inside cancer cells. Herein, we report an efficient lysosome-controlled ROS overproduction via a pH-responsive catalytic nanosystem (FeOx-MSNs), which catalyze the decomposition of H2O2 to produce considerable ROS selectively inside the acidic lysosomes (pH 5.0) of cancer cells. After a further incorporation of ROS-sensitive TMB into the nanosystem (FeOx-MSNs-TMB), both a distinct cell labeling and an efficient death of breast carcinoma cells are obtained. This lysosome-controlled efficient ROS overproduction suggests promising applications in cancer treatments.Excess reactive oxygen species (ROS) have been proved to damage cancer cells efficiently. ROS overproduction is thus greatly desirable for cancer therapy. To date, ROS production is generally uncontrollable and outside cells, which always bring severe side-effects in the vasculature. Since most ROS share a very short half-life and primarily react close to their site of formation, it would be more efficient if excess ROS are controllably produced inside cancer cells. Herein, we report an efficient lysosome-controlled ROS overproduction via a pH-responsive catalytic nanosystem (FeOx-MSNs), which catalyze the decomposition of H2O2 to produce considerable ROS selectively inside the acidic lysosomes (pH 5.0) of cancer cells. After a further incorporation of ROS-sensitive TMB into the nanosystem (FeOx-MSNs-TMB), both a distinct cell labeling and an efficient death of breast carcinoma cells are obtained. This lysosome-controlled efficient ROS overproduction suggests promising applications in cancer treatments. Electronic supplementary information (ESI) available: Experimental section, supplementary figures and characterization of as-prepared compounds. See DOI: 10.1039/c5nr00706b

  19. Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars

    PubMed Central

    Cavanagh, Colin R.; Chao, Shiaoman; Wang, Shichen; Huang, Bevan Emma; Stephen, Stuart; Kiani, Seifollah; Forrest, Kerrie; Saintenac, Cyrille; Brown-Guedira, Gina L.; Akhunova, Alina; See, Deven; Bai, Guihua; Pumphrey, Michael; Tomar, Luxmi; Wong, Debbie; Kong, Stephan; Reynolds, Matthew; da Silva, Marta Lopez; Bockelman, Harold; Talbert, Luther; Anderson, James A.; Dreisigacker, Susanne; Baenziger, Stephen; Carter, Arron; Korzun, Viktor; Morrell, Peter Laurent; Dubcovsky, Jorge; Morell, Matthew K.; Sorrells, Mark E.; Hayden, Matthew J.; Akhunov, Eduard

    2013-01-01

    Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat. PMID:23630259

  20. ?2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes*

    PubMed Central

    Jakhria, Toral; Hellewell, Andrew L.; Porter, Morwenna Y.; Jackson, Matthew P.; Tipping, Kevin W.; Xue, Wei-Feng; Radford, Sheena E.; Hewitt, Eric W.

    2014-01-01

    Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of ?2-microglobulin (?2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented ?2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented ?2m fibrils did not, however, cause cell death. Instead, fragmented ?2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway. PMID:25378395

  1. 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

  2. Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity.

    PubMed

    Guan, Jian; Mishra, Shikha; Qiu, Yiling; Shi, Jianru; Trudeau, Kyle; Las, Guy; Liesa, Marc; Shirihai, Orian S; Connors, Lawreen H; Seldin, David C; Falk, Rodney H; MacRae, Calum A; Liao, Ronglih

    2014-11-01

    AL amyloidosis is the consequence of clonal production of amyloidogenic immunoglobulin light chain (LC) proteins, often resulting in a rapidly progressive and fatal amyloid cardiomyopathy. Recent work has found that amyloidogenic LC directly initiate a cardio-toxic response underlying the pathogenesis of the cardiomyopathy; however, the mechanisms that contribute to this proteotoxicity remain unknown. Using human amyloidogenic LC isolated from patients with amyloid cardiomyopathy, we reveal that dysregulation of autophagic flux is critical for mediating amyloidogenic LC proteotoxicity. Restoration of autophagic flux by pharmacological intervention using rapamycin protected against amyloidogenic light chain protein-induced pathologies including contractile dysfunction and cell death at the cellular and organ level and also prolonged survival in an in vivo zebrafish model of amyloid cardiotoxicity. Mechanistically, we identify impaired lysosomal function to be the major cause of defective autophagy and amyloidogenic LC-induced proteotoxicity. Collectively, these findings detail the downstream molecular mechanisms underlying AL amyloid cardiomyopathy and highlight potential targeting of autophagy and lysosomal dysfunction in patients with amyloid cardiomyopathy. PMID:25319546

  3. Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway? †

    PubMed Central

    Varghese, Bentley; Barriere, Herve; Carbone, Christopher J.; Banerjee, Anamika; Swaminathan, Gayathri; Plotnikov, Alexander; Xu, Ping; Peng, Junmin; Goffin, Vincent; Lukacs, Gergely L.; Fuchs, Serge Y.

    2008-01-01

    The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling. PMID:18573876

  4. Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

    SciTech Connect

    Mithieux, G.; Rousset, B.

    1989-03-15

    We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells.

  5. Lysosomal localization of Japanese medaka (Oryzias latipes) Neu1 sialidase and its highly conserved enzymatic profiles with human.

    PubMed

    Ryuzono, Sena; Takase, Ryo; Oishi, Kazuki; Ikeda, Asami; Chigwechokha, Petros Kingstone; Funahashi, Aki; Komatsu, Masaharu; Miyagi, Taeko; Shiozaki, Kazuhiro

    2016-01-10

    Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase. PMID:26432003

  6. 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.

  7. The pan erbB inhibitor PD168393 enhances lysosomal dysfunction-induced apoptotic death in malignant peripheral nerve sheath tumor cells

    PubMed Central

    Kohli, Latika; Kaza, Niroop; Lavalley, Nicholas J.; Turner, Kathryn L.; Byer, Stephanie; Carroll, Steven L.; Roth, Kevin A.

    2012-01-01

    Malignant peripheral nerve sheath tumors (MPNSTs) are rapidly progressive Schwann cell neoplasms. The erbB family of membrane tyrosine kinases has been implicated in MPNST mitogenesis and invasion and, thus, is a potential therapeutic target. However, tyrosine kinase inhibitors (TKIs) used alone have limited tumoricidal activity. Manipulating the autophagy lysosomal pathway in cells treated with cytostatic agents can promote apoptotic cell death in some cases. The goal of this study was to establish a mechanistic basis for formulating drug combinations to effectively trigger death in MPNST cells. We assessed the effects of the pan erbB inhibitor PD168393 on MPNST cell survival, caspase activation, and autophagy. PD168393 induced a cytostatic but not a cytotoxic response in MPNST cells that was accompanied by suppression of Akt and mTOR activation and increased autophagic activity. The effects of autophagy modulation on MPNST survival were then assessed following the induction of chloroquine (CQ)–induced lysosomal stress. In CQ-treated cells, suppression of autophagy was accompanied by increased caspase activation. In contrast, increased autophagy induction by inhibition of mTOR did not trigger cytotoxicity, possibly because of Akt activation. We thus hypothesized that dual targeting of mTOR and Akt by PD168393 would significantly increase cytotoxicity in cells exposed to lysosomal stress. We found that PD168393 and CQ in combination significantly increased cytotoxicity. We conclude that combinatorial therapies with erbB inhibitors and agents inducing lysosomal dysfunction may be an effective means of treating MPNSTs. PMID:22259051

  8. K-Targeted Metabolomic Analysis Extends Chemical Subtraction to DESIGNER Extracts: Selective Depletion of Extracts of Hops (Humulus lupulus)?

    PubMed Central

    2015-01-01

    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 1H NMR, LC-MS, and HiFSA-based NMR fingerprinting. PMID:25437744

  9. 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

  10. The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

    PubMed

    Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

    2015-04-10

    Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1? yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1? vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1? vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1? or vph1? fab1? double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes. PMID:25713145

  11. Crossreactive ?? T Cell Receptors Are the Predominant Targets of Thymocyte Negative Selection.

    PubMed

    McDonald, Benjamin D; Bunker, Jeffrey J; Erickson, Steven A; Oh-Hora, Masatsugu; Bendelac, Albert

    2015-11-17

    The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual pre-selection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4(-)CD8?(-) intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation. PMID:26522985

  12. Rab25 and CLIC3 Collaborate to Promote Integrin Recycling from Late Endosomes/Lysosomes and Drive Cancer Progression

    PubMed Central

    Dozynkiewicz, Marta A.; Jamieson, Nigel B.; MacPherson, Iain; Grindlay, Joan; van den Berghe, Peter V.E.; von Thun, Anne; Morton, Jennifer P.; Gourley, Charlie; Timpson, Paul; Nixon, Colin; McKay, Colin J.; Carter, Ross; Strachan, David; Anderson, Kurt; Sansom, Owen J.; Caswell, Patrick T.; Norman, Jim C.

    2012-01-01

    Summary Here we show that Rab25 permits the sorting of ligand-occupied, active-conformation ?5?1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active ?5?1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo. PMID:22197222

  13. Selective targeting of PPAR? by the natural product chelerythrine with a unique binding mode and improved antidiabetic potency

    PubMed Central

    Zheng, Weili; Qiu, Lin; Wang, Rui; Feng, Xuhui; Han, Yaping; Zhu, Yanlin; Chen, Dezhou; Liu, Yijie; Jin, Lihua; Li, Yong

    2015-01-01

    Type 2 diabetes mellitus (T2DM) is a pervasive metabolic syndrome that is characterized by insulin resistance, hyperglycemia and dyslipidemia. As full agonists of PPAR?, thiazolidinedione (TZD) drugs elicit antidiabetic effects by targeting PPAR? but is accompanied by weight gain, fluid retention and cardiovascular risk associated with their transcriptional agonism potency. We here identify a natural product chelerythrine as a unique selective PPAR modulator (SPPARM) with a potent PPAR? binding activity but much less classical receptor transcriptional agonism. Structural analysis reveals that chelerythrine exhibits unique binding in parallel with H3 of PPAR?. Unlike TZDs, chelerythrine destabilizes helix 12, especially residue tyrosine 473, resulting in a loose configuration of AF-2 and a selective cofactor profile distinct from TZDs, leading to a differential target gene profile in adipogenesis in db/db diabetic mice. Moreover, chelerythrine improved insulin sensitivity by more potently blocking the phosphorylation of PPAR? by CDK5 compared to TZDs. These data fundamentally elucidate the mechanism by which chelerythrine retains the benefits of improving insulin sensitivity while reducing the adverse effects of TZDs, suggesting that the natural product chelerythrine is a very promising pharmacological agent by selectively targeting PPAR? for further development in the clinical treatment of insulin resistance. PMID:26183621

  14. Altered TFEB-mediated lysosomal biogenesis in Gaucher disease iPSC-derived neuronal cells.

    PubMed

    Awad, Ola; Sarkar, Chinmoy; Panicker, Leelamma M; Miller, Diana; Zeng, Xianmin; Sgambato, Judi A; Lipinski, Marta M; Feldman, Ricardo A

    2015-10-15

    Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase). The severe forms of GD are associated with neurodegeneration with either rapid (Type 2) or slow progression (Type 3). Although the neurodegenerative process in GD has been linked to lysosomal dysfunction, the mechanisms involved are largely unknown. To identify the lysosomal alterations in GD neurons and uncover the mechanisms involved, we used induced pluripotent stem cells (iPSCs) derived from patients with GD. In GD iPSC-derived neuronal cells (iPSC-NCs), GBA1 mutations caused widespread lysosomal depletion, and a block in autophagic flux due to defective lysosomal clearance of autophagosomes. Autophagy induction by rapamycin treatment in GD iPSC-NCs led to cell death. Further analysis showed that in GD iPSC-NCs, expression of the transcription factor EB (TFEB), the master regulator of lysosomal genes, and lysosomal gene expression, were significantly downregulated. There was also reduced stability of the TFEB protein and altered lysosomal protein biosynthesis. Treatment of mutant iPSC-NCs with recombinant GCase (rGCase) reverted the lysosomal depletion and autophagy block. The effect of rGCase on restoring lysosomal numbers in mutant cells was enhanced in the presence of overexpressed TFEB, but TFEB overexpression alone did not reverse the lysosomal depletion phenotype. Our results suggest that GBA1 mutations interfere with TFEB-mediated lysosomal biogenesis, and that the action of GCase in maintaining a functioning pool of lysosomes is exerted in part through TFEB. The lysosomal alterations described here are likely to be a major determinant in GBA1-associated neurodegeneration. PMID:26220978

  15. Lysosomal membrane damage in soluble Abeta-mediated cell death in Alzheimer's disease.

    PubMed

    Ditaranto, K; Tekirian, T L; Yang, A J

    2001-02-01

    Our previous studies suggest that a failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes is a mechanism that contributes to intracellular accumulation in Alzheimer's disease. In this study, we demonstrate that cultured primary neurons are able to internalize soluble Abeta1-42 from the culture medium and accumulate inside the endosomal/lysosomal system. The intracellular Abeta1-42 is resistant to protease degradation and stable for at least 48 h within the cultured neurons. Incubation of cultured neurons with a cytotoxic concentration of soluble Abeta1-42 invokes the rapid free radical generation within lysosomes and disruption of lysosomal membrane proton gradient which precedes cell death. The loss of lysosomal membrane impermeability is only specific to the Abeta1-42 isoform since incubation of cells with high concentrations of Abeta1-40 has no effect on lysosomal hydrolase release. To further support the role of lysosomal membrane damage in Abeta-mediated cell death, we demonstrate that photodisruption of acridine orange (AO)-loaded lysosomes with intense blue light induces a relatively rapid synchronous lysosomal membrane damage and neuronal death similar to that observed as a result of Abeta exposure. AO leaks quickly from late endosomes and lysosomes and partially shifts the fluorescence from an orange fluorescence to a diffuse, green cytoplasmic fluorescence. Such AO relocalization is due to an initial disruption of the lysosomal proton gradient, followed by the release of lysosomal hydrolases into the cytoplasmic compartment. Treatment of cells with either the antioxidant n-propyl gallate or lysosomotropic amine (methylamine) partially blocks the release of lysosomal contents suggesting that this AO relocalization is due to lysosomal membrane oxidation. Based on these findings, we propose that the cell death mediated by the soluble Abeta may be fundamentally different from the cell loss observed following extracellular Abeta deposition. PMID:11162237

  16. Target Selection for SETI. II. Tycho-2 Dwarfs, Old Open Clusters, and the Nearest 100 Stars

    NASA Astrophysics Data System (ADS)

    Turnbull, Margaret C.; Tarter, Jill C.

    2003-12-01

    We present the full target list and prioritization algorithm developed for use by the microwave search for technological signals at the SETI Institute. We have included the Catalog of Nearby Habitable Stellar Systems (HabCat, described in Paper I), all of the nearest 100 stars and 14 old open clusters. This is further augmented by a subset of the Tycho-2 catalog based on reduced proper motions, and this larger catalog should routinely provide at least three target stars within the large primary field of view of the Allen Telescope Array. The algorithm for prioritizing objects in the full target list includes scoring based on the subset category of each target (i.e., HabCat, cluster, Tycho-2, or nearest 100), its distance (if known), and its proximity to the Sun on the color-magnitude diagram.

  17. 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 ...

  18. 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 ...

  19. 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

  20. Gaucher's disease: the changing paradigm of a lysosomal disorder.

    PubMed

    Mehta, Atul

    2011-09-01

    Gaucher's disease (GD) is the most common lysosomal storage disease with a frequency of approximately 1:50,000 people. It is the result of the deficiency of the lysosomal enzyme beta-glucocerebrosidase. The deficiency of the enzyme results in the accumulation of the substrate, glucosyl-ceramide, in the organs. Substitutive enzymatic treatment has been available since almost 20 years. This brief overview highlights some of the most important milestones and the treatments for this disease. The study of this rare disorder is beginning to provide information on the pathogenesis of common diseases such as Parkinson's disease or cancer. Individuals with GD are at greater risk of developing cancer in general, especially hepatobiliary and hematologic (multiple myeloma and B-cell neoplasms). This association has been attributed to the immunologic abnormalities associated with abnormal expression of cytokines such as interleukin-6. Alternative and complementary, some recently marketed and licensed, are providing options for patients throughout Europe and the world. PMID:22230118

  1. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    PubMed Central

    Petters, Edyta; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2015-01-01

    Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer. PMID:26307975

  2. SDSS-III Baryon Oscillation Spectroscopic Survey Data Release 12: galaxy target selection and large-scale structure catalogues

    NASA Astrophysics Data System (ADS)

    Reid, Beth; Ho, Shirley; Padmanabhan, Nikhil; Percival, Will J.; Tinker, Jeremy; Tojeiro, Rita; White, Martin; Eisenstein, Daniel J.; Maraston, Claudia; Ross, Ashley J.; Sánchez, Ariel G.; Schlegel, David; Sheldon, Erin; Strauss, Michael A.; Thomas, Daniel; Wake, David; Beutler, Florian; Bizyaev, Dmitry; Bolton, Adam S.; Brownstein, Joel R.; Chuang, Chia-Hsun; Dawson, Kyle; Harding, Paul; Kitaura, Francisco-Shu; Leauthaud, Alexie; Masters, Karen; McBride, Cameron K.; More, Surhud; Olmstead, Matthew D.; Oravetz, Daniel; Nuza, Sebastián E.; Pan, Kaike; Parejko, John; Pforr, Janine; Prada, Francisco; Rodríguez-Torres, Sergio; Salazar-Albornoz, Salvador; Samushia, Lado; Schneider, Donald P.; Scóccola, Claudia G.; Simmons, Audrey; Vargas-Magana, Mariana

    2016-01-01

    The Baryon Oscillation Spectroscopic Survey (BOSS), part of the Sloan Digital Sky Survey (SDSS) III project, has provided the largest survey of galaxy redshifts available to date, in terms of both the number of galaxy redshifts measured by a single survey, and the effective cosmological volume covered. Key to analysing the clustering of these data to provide cosmological measurements is understanding the detailed properties of this sample. Potential issues include variations in the target catalogue caused by changes either in the targeting algorithm or properties of the data used, the pattern of spectroscopic observations, the spatial distribution of targets for which redshifts were not obtained, and variations in the target sky density due to observational systematics. We document here the target selection algorithms used to create the galaxy samples that comprise BOSS. We also present the algorithms used to create large-scale structure catalogues for the final Data Release (DR12) samples and the associated random catalogues that quantify the survey mask. The algorithms are an evolution of those used by the BOSS team to construct catalogues from earlier data, and have been designed to accurately quantify the galaxy sample. The code used, designated MKSAMPLE, is released with this paper.

  3. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    NASA Astrophysics Data System (ADS)

    di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-11-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3?-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

  4. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    PubMed Central

    Di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-01-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3?-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation. PMID:26567894

  5. Structure of human saposin A at lysosomal pH

    PubMed Central

    Hill, Chris H.; Read, Randy J.; Deane, Janet E.

    2015-01-01

    The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme ?-galactocerebrosidase (GALC), which catalyzes the breakdown of ?-d-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a ‘closed’ to an ‘open’ conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8?Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined ‘closed’ conformation, showing that pH alone is not sufficient for the transition to the ‘open’ conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility. PMID:26144235

  6. Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

    PubMed

    Hermans, M M; Kroos, M A; van Beeumen, J; Oostra, B A; Reuser, A J

    1991-07-25

    The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function. PMID:1856189

  7. Treatment of lysosomal storage disorders: successes and challenges.

    PubMed

    Hollak, Carla E M; Wijburg, Frits A

    2014-07-01

    Treatment options for a number of lysosomal storage disorders have rapidly expanded and currently include enzyme replacement therapy, substrate reduction, chaperone treatment, hematopoietic stem cell transplantation, and gene-therapy. Combination treatments are also explored. Most therapies are not curative but change the phenotypic expression of the disease. The effectiveness of treatment varies considerably between the different diseases, but also between sub-groups of patients with a specific lysosomal storage disorder. The heterogeneity of the patient populations complicates the prediction of benefits of therapy, specifically in patients with milder disease manifestations. In addition, there is a lack of data on the natural history of diseases and disease phenotypes. Initial trial data show benefits on relevant short-term endpoints, but the real world situation may reveal different outcomes. Collaborative international studies are much needed to study the long-term clinical efficacy of treatments, and to detect new complications or associated conditions of the diseases. This review summarizes the available treatment modalities for lysosomal storage disorders and the challenges associated with long term clinical care for these patients. PMID:24820227

  8. Economic assessment of FEC-based targeted selective drenching in horses.

    PubMed

    Sallé, Guillaume; Cortet, Jacques; Koch, Christine; Reigner, Fabrice; Cabaret, Jacques

    2015-11-30

    In the face of an increased prevalence of drug-resistant cyathostomin populations, a targeted selective treatment (TST) strategy based on Faecal Egg Counts (FECs) has been proposed as an alternative management strategy. However, associated costs may be a barrier to the uptake of this strategy. Our study aims to provide an economic assessment of FEC-based TST. FECs were determined in a Welsh pony herd thrice a year from 2010 to 2014. This database was used to explore the impact of FEC price, sampling strategy (individual or pooled) and labour-associated costs. Drug price was set at the cheapest level, hence providing a conservative framework to determine the maximum viable FEC price in the context of a cost-driven horse industry. The maximum viable FEC price for a cost-efficient individual based strategy was determined by an in silico bootstrap approach consisting of randomly sampling 1000 virtual pony herds of various sizes (1 to 100 ponies) from the available database and estimating the associated costs (FEC price ranging from €1 to €10, anthelmintic costs and labour-associated costs). The costs and benefits of the pooling strategy that consists of basing the decision to treat on group FEC values were also investigated. This is thought to reduce FEC-based costs but may result in highly infected individuals being left undrenched, i.e. in false-negatives, as a result of FEC overdispersion. For various pool-sizes (1-20 ponies) and various cut-off thresholds (50-200eggs/g), we sampled 1000 pony herds in silico to estimate the associated costs and determine the number of positive ponies within a negative pool. Following these simulations, pool-based FECs of various sizes were performed on 40 ponies to compare predictions with real data. Within 4 years, anthelmintic costs were cut by 80%, albeit with free FECs. In silico estimations suggested that an individual FEC-based TST would not be cost-efficient in this context for an FEC price above €5. With a pooled FEC strategy, the proportion of false-negatives never exceeded 15% of the pool size. The combination of a 14-pony pool and a cut-off value of 150eggs/g minimized total costs while keeping the number of false negatives to a reasonable level. Real data obtained from infected ponies however, suggested that pool size should not exceed 10 individuals, since the inhomogeneous mixing of faeces of larger pools probably reduced the correlation between average pooled FECs and the mean of individual FECs. Our study provides an economic framework that could be valuable for emphasizing the use of FEC-based approaches in the field. PMID:26414907

  9. Beam Normal Single Spin Asymmetries in Elastic Electron Scattering from Selected Targets

    NASA Astrophysics Data System (ADS)

    Duvall, Wade; Qweak Collaboration

    2013-10-01

    The primary focus of the Qweak experiment at Jefferson Lab is the determination of the proton's weak charge. To make corrections to the measured asymmetry, dedicated measurements were made of the parity-conserving beam normal single spin asymmetries (An) in elastic scattering of transversly polarized electrons from several unpolarized targets. The targets included hydrogen, carbon, and aluminum. In the case of hydrogen, two separate spectrometer settings were used to study elastic scattering off both the protons and electrons in the hydrogen target. The An for the hadronic targets are dominated by two-photon exchange amplitudes, which were important in resolving the discrepancy between two different methods determining the proton's electromagnetic form factors. For the heavy nuclear targets, proper calculation of Coulomb distortion effects are also important. For the case of the elastic scattering from electrons (Moller scattering), the asymmetry is a calculable QED process. The measurements were made with the Qweak apparatus at a beam energy of 1165 MeV and central scattering angle of ~8° . An overview of the motivations, the experimental approach, and the status of the analysis will be presented. Supported in part by NSF Award PHY1101818.

  10. Inhibition of C-terminal binding protein attenuates transcription factor 4 signaling to selectively target colon cancer stem cells

    PubMed Central

    Patel, Jagrut; Baranwal, Somesh; Love, Ian M; Patel, Nirmita J; Grossman, Steven R; Patel, Bhaumik B

    2014-01-01

    Selective targeting of cancer stem cells (CSCs), implicated in tumor relapse, holds great promise in the treatment of colorectal cancer. Overexpression of C-terminal binding protein (CtBP), an NADH dependent transcriptional regulator, is often observed in colon cancer. Of note, TCF-4 signaling is also up-regulated in colonic CSCs. We hypothesized that CtBP, whose dehydrogenase activity is amenable to pharmacological inhibition by 4-methylthio-2-oxobutyric acid (MTOB), positively regulates TCF-4 signaling, leading to CSC growth and self-renewal. CSCs demonstrated significant upregulation of CtBP1 and CtBP2 levels (mRNA and protein) and activity partly due to increased NADH/NAD ratio, as well as increased TCF/LEF transcriptional activity, compared to respective controls. Depletion of CtBP2 inhibited, while its overexpression enhanced, CSC growth (1° spheroids) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of spheroid growth and self-renewal in a dose dependent manner. MTOB displayed significantly greater selectivity for growth inhibition in the spheroids, at least in part through induction of apoptosis, compared to monolayer controls. Moreover, MTOB inhibited basal as well as induced (by GSK-3? inhibitor) TCF/LEF activity while suppressing mRNA and protein levels of several ?-catenin target genes (CD44, Snail, C-MYC and LGR5). Lastly, CtBP physically interacted with TCF-4, and this interaction was significantly inhibited in the presence of MTOB. The above findings point to a novel role of CtBPs in the promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a novel approach for treatment of colorectal cancer focused on targeting of CSCs. PMID:25483087

  11. Design of an EGFR-targeting toxin for photochemical delivery: in vitro and in vivo selectivity and efficacy.

    PubMed

    Berstad, M B; Cheung, L H; Berg, K; Peng, Q; Fremstedal, A S V; Patzke, S; Rosenblum, M G; Weyergang, A

    2015-10-29

    The number of epidermal growth factor receptor (EGFR)-targeting drugs in the development for cancer treatment is continuously increasing. Currently used EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors have specific limitations related to toxicity and development of resistance, and there is a need for alternative treatment strategies to maximize the clinical potential of EGFR as a molecular target. This study describes the design and production of a novel EGFR-targeted fusion protein, rGel/EGF, composed of the recombinant plant toxin gelonin and EGF. rGel/EGF was custom-made for administration by photochemical internalization (PCI), a clinically tested modality for cytosolic release of macromolecular therapeutics. rGel/EGF lacks efficient mechanisms for endosomal escape and is therefore minimally toxic as monotherapy. However, PCI induces selective and efficient cytosolic release of rGel/EGF in EGFR-expressing target cells by light-directed activation of photosensitizers accumulated selectively in tumor tissue. PCI of rGel/EGF was shown to be highly effective against EGFR-expressing cell lines, including head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (Erbitux). Apoptosis, necrosis and autophagy were identified as mechanisms of action following PCI of rGel/EGF in vitro. PCI of rGel/EGF was further shown as a highly tumor-specific and potent modality in vivo, with growth inhibitory effects demonstrated on A-431 squamous cell carcinoma (SCC) xenografts and reduction of tumor perfusion and necrosis induction in SCC-026 HNSCC tumors. Considering the small amount of rGel/EGF injected per animal (0.1?mg/kg), the presented in vivo results are highly promising and warrant optimization and production of rGel/EGF for further preclinical evaluation with PCI. PMID:25684137

  12. 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.

  13. Iterative Time Reversal Simulation for Selective Focusing in Multi-target Nonlinear Media

    NASA Astrophysics Data System (ADS)

    Su, Chang; Peng, Zhefan; Lin, Weijun

    In High Intensity Focused Ultrasound (HIFU), when multiple targets are present in a linear medium, ultrasound can focus on the strongest target by using an iterative time-reversal(TR) method. However, the validation of iterative TR in nonlinear human tissue still needs to be investigated. In the study, the TR and iterative TR processes are numerically simulated with a finite difference method in two dimension, considering the nonlinear effects. Results show that TR is valid in nonlinear human tissues with some difference in focus accuracy and intensity gain comparing to that in linear media. The nonlinearity of the media increases the intensity gain at the focal point, while the absorption decreases the focal gain and changes the position of the focal spot. Iterative TR works well in nonlinear media and the lobe on the weaker target attenuates more rapidly than in linear media.

  14. ERP markers of target selection discriminate children with high vs. low working memory capacity

    PubMed Central

    Shimi, Andria; Nobre, Anna Christina; Scerif, Gaia

    2015-01-01

    Selective attention enables enhancing a subset out of multiple competing items to maximize the capacity of our limited visual working memory (VWM) system. Multiple behavioral and electrophysiological studies have revealed the cognitive and neural mechanisms supporting adults’ selective attention of visual percepts for encoding in VWM. However, research on children is more limited. What are the neural mechanisms involved in children’s selection of incoming percepts in service of VWM? Do these differ from the ones subserving adults’ selection? Ten-year-olds and adults used a spatial arrow cue to select a colored item for later recognition from an array of four colored items. The temporal dynamics of selection were investigated through EEG signals locked to the onset of the memory array. Both children and adults elicited significantly more negative activity over posterior scalp locations contralateral to the item to-be-selected for encoding (N2pc). However, this activity was elicited later and for longer in children compared to adults. Furthermore, although children as a group did not elicit a significant N2pc during the time-window in which N2pc was elicited in adults, the magnitude of N2pc during the “adult time-window” related to their behavioral performance during the later recognition phase of the task. This in turn highlights how children’s neural activity subserving attention during encoding relates to better subsequent VWM performance. Significant differences were observed when children were divided into groups of high vs. low VWM capacity as a function of cueing benefit. Children with large cue benefits in VWM capacity elicited an adult-like contralateral negativity following attentional selection of the to-be-encoded item, whereas children with low VWM capacity did not. These results corroborate the close coupling between selective attention and VWM from childhood and elucidate further the attentional mechanisms constraining VWM performance in children. PMID:26594157

  15. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    SciTech Connect

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-02-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

  16. TFEB activation promotes the recruitment of lysosomal glycohydrolases ?-hexosaminidase and ?-galactosidase to the plasma membrane

    SciTech Connect

    Magini, Alessandro; Department of Medical and Biological Sciences , University of Udine, Udine ; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

    2013-10-18

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases ?-hexosaminidase and ?-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

  17. A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural products.

    PubMed

    Liang, Junling; Meng, Jie; Wu, Dingfang; Guo, Mengzhe; Wu, Shihua

    2015-06-26

    Counter-current chromatography (CCC) is an efficient liquid-liquid chromatography technique for separation and purification of complex mixtures like natural products extracts and synthetic chemicals. However, CCC is still a challenging process requiring some special technical knowledge especially in the selection of appropriated solvent systems. In this work, we introduced a new 9 × 9 map-based solvent selection strategy for CCC isolation of targets, which permit more than 60 hexane-ethyl acetate-methanol-water (HEMWat) solvent systems as the start candidates for the selection of solvent systems. Among these solvent systems, there are clear linear correlations between partition coefficient (K) and the system numbers. Thus, an appropriate CCC solvent system (i.e., sweet spot for K = 1) may be hit by measurement of k values of the target only in two random solvent systems. Besides this, surprisingly, we found that through two sweet spots, we could get a line ("Sweet line") where there are infinite sweet solvent systems being suitable for CCC separation. In these sweet solvent systems, the target has the same partition coefficient (K) but different solubilities. Thus, the better sweet solvent system with higher sample solubility can be obtained for high capacity CCC preparation. Furthermore, we found that there is a zone ("Sweet zone") where all solvent systems have their own sweet partition coefficients values for the target in range of 0.4 < K< 2.5 or extended range of 0.25 < K < 16. All results were validated by using 14 pure GUESSmix mimic natural products as standards and further confirmed by isolation of several targets including honokiol and magnolol from the extracts of Magnolia officinalis Rehd. Et Wils and tanshinone IIA from Salvia miltiorrhiza Bunge. In practice, it is much easier to get a suitable solvent system only by making a simple screening two to four HEMWat two-phase solvent systems to obtain the sweet line or sweet zone without special knowledge or comprehensive standards as references. This is an important advancement for solvent system selection and also will be very useful for isolation of current natural products including Traditional Chinese Medicines. PMID:25980692

  18. Highly sensitive and selective analysis of widely targeted metabolomics using gas chromatography/triple-quadrupole mass spectrometry.

    PubMed

    Tsugawa, Hiroshi; Tsujimoto, Yuki; Sugitate, Kuniyo; Sakui, Norihiro; Nishiumi, Shin; Bamba, Takeshi; Fukusaki, Eiichiro

    2014-01-01

    In metabolomics studies, gas chromatography coupled with time-of-flight or quadrupole mass spectrometry has frequently been used for the non-targeted analysis of hydrophilic metabolites. However, because the analytical platform employs the deconvolution method to extract single-metabolite information from co-eluted peaks and background noise, the extracted peak is artificial product depending on the mathematical parameters and is not completely compatible with the pure component obtained by analyzing a standard compound. Moreover, it has insufficient ability for quantitative metabolomics. Therefore, highly sensitive and selective methods capable of pure peak extraction without any complicated mathematical techniques are needed. For this purpose, we have developed a novel analytical method using gas chromatography coupled with triple-quadrupole mass spectrometry (GC-QqQ/MS). We developed a selected reaction monitoring (SRM) method to analyze the trimethylsilyl derivatives of 110 metabolites, using electron ionization. This methodology enables us to utilize two complementary techniques-non-targeted and widely targeted metabolomics in the same sample preparation protocol, which would facilitate the formulation or verification of novel hypotheses in biological sciences. The GC-QqQ/MS analysis can accurately identify a metabolite using multichannel SRM transitions and intensity ratios in the analysis of living organisms. In addition, our methodology offers a wide dynamic range, high sensitivity, and highly reproducible metabolite profiles, which will contribute to the biomarker discoveries and quality evaluations in biology, medicine, and food sciences. PMID:23867096

  19. Selective targeting of nuclear receptor FXR by avermectin analogues with therapeutic effects on nonalcoholic fatty liver disease

    PubMed Central

    Jin, Lihua; Wang, Rui; Zhu, Yanlin; Zheng, Weili; Han, Yaping; Guo, Fusheng; Ye, Frank Bin; Li, Yong

    2015-01-01

    Non-alcoholic fatty liver disease (NAFLD) has become a predictive factor of death from many diseases. Farnesoid X receptor (FXR) is an ideal target for NAFLD drug development due to its crucial roles in lipid metabolism. The aim of this work is to examine the molecular mechanisms and functional roles of FXR modulation by avermectin analogues in regulating metabolic syndromes like NAFLD. We found that among avermectin analogues studied, the analogues that can bind and activate FXR are effective in regulating metabolic parameters tested, including reducing hepatic lipid accumulation, lowering serum cholesterol and glucose levels, and improving insulin sensitivity, in a FXR dependent manner. Mechanistically, the avermectin analogues that interact with FXR exhibited features as partial agonists, with distinctive properties in modulating coregulator recruitment. Structural features critical for avermectin analogues to selectively bind to FXR were also revealed. This study indicated that in addition to antiparasitic activity, avermectin analogues are promising drug candidates to treat metabolism syndrome including NAFLD by directly targeting FXR. Additionally, the structural features that discriminate the selective binding of FXR by avermectin analogues may provide a unique safe approach to design drugs targeting FXR signaling. PMID:26620317

  20. 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…

  1. Mitochondrial targeting of electron scavenging antioxidants: Regulation of selective oxidation vs random chain reactions

    PubMed Central

    Kagan, Valerian E.; Wipf, Peter; Stoyanovsky, Detcho; Greenberger, Joel S.; Borisenko, Grigory; Belikova, Natalia A.; Yanamala, Naveena; Samhan Arias, Alejandro K.; Tungekar, Muhammad A.; Jiang, Jianfei; Tyurina, Yulia Y.; Ji, Jing; Klein-Seetharaman, Judith; Pitt, Bruce R.; Shvedova, Anna A; Bay?r, Hülya

    2009-01-01

    Effective regulation of highly compartmentalized production of reactive oxygen species and peroxidation reactions in mitochondria requires targeting of small molecule antioxidants and antioxidant enzymes into the organelles. This review describes recently developed approaches to mitochondrial targeting of small biologically active molecules based on: (i) preferential accumulation in mitochondria because of their hydrophobicity and positive charge (hydrophobic cations), (ii) binding with high affinity to an intra-mitochondrial constituent, and (iii) metabolic conversions by specific mitochondrial enzymes to reveal an active entity. In addition, targeted delivery of antioxidant enzymes via expression of leader-sequences directing the proteins into mitochondria is considered. Examples of successful antioxidant and anti-apoptotic protection based on the ability of targeted cargoes to inhibit cytochrome c-catalyzed peroxidation of a mitochondria-specific phospholipid cardiolipin, in vitro and in vivo are presented. Particular emphasis is placed on the employment of triphenylphosphonium- and hemi-gramicidin S-moieties as two effective vehicles for mitochondrial delivery of antioxidants. PMID:19716396

  2. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte

    E-print Network

    Lieberman, Judy

    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 potential immunosuppressive effects on bystander lymphocytes. However, primary lympho- cytes are highly

  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. A combinatorial systematic evolution of ligands by exponential enrichment method for selection of aptamer against protein targets.

    PubMed

    Mondal, Bhairab; Ramlal, Shylaja; Lavu, Padma Sudha Rani; Murali, Harishchandra Sreepathy; Batra, Harsh Vardhan

    2015-11-01

    Aptamers are synthetic DNA recognition elements which form unique conformations that enable them to bind specifically to their targets. In the present study, an attempt was made to standardize a new modified combinatorial method comprising of Ni-NTA affinity Systematic Evolution of Ligands by Exponential Enrichment (SELEX; based on affinity between His tag protein and Ni-NTA), membrane SELEX (based on immobilization of protein on nitrocellulose membrane), and microtiter plate based SELEX (to monitor affinity and to enrich the selected aptamers) for protein targets. For experimental evaluation, staphylococcal interotoxin B was the molecule chosen. The new combinatorial method enhanced selection ability up to 51.20 % in comparison with individual conventional procedures. Employing this method following six rounds of selection, high-affinity aptamers with very different properties could be obtained with a dissociation constant (K d) value as low as 34.72?±?25.09 nM. The optimal aptamers could be employed in fluorescence binding assay, enzyme-linked oligonucleotide assays, and aptamer-based Western blot assay for characterization and detection. These results pave a potential path without using of any robotics for high-throughput generation of aptamers with advantages in terms of rapidity, simplicity, and ease in handling. PMID:26293334

  6. Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates nephrogenic diabetes insipidus.

    PubMed

    Olesen, Emma T B; Rützler, Michael R; Moeller, Hanne B; Praetorius, Helle A; Fenton, Robert A

    2011-08-01

    In the kidney, the actions of vasopressin on its type-2 receptor (V2R) induce increased water reabsorption alongside polyphosphorylation and membrane targeting of the water channel aquaporin-2 (AQP2). Loss-of-function mutations in the V2R cause X-linked nephrogenic diabetes insipidus. Treatment of this condition would require bypassing the V2R to increase AQP2 membrane targeting, but currently no specific pharmacological therapy is available. The present study examined specific E-prostanoid receptors for this purpose. In vitro, prostaglandin E2 (PGE2) and selective agonists for the E-prostanoid receptors EP2 (butaprost) or EP4 (CAY10580) all increased trafficking and ser-264 phosphorylation of AQP2 in Madin-Darby canine kidney cells. Only PGE2 and butaprost increased cAMP and ser-269 phosphorylation of AQP2. Ex vivo, PGE2, butaprost, or CAY10580 increased AQP2 phosphorylation in isolated cortical tubules, whereas PGE2 and butaprost selectively increased AQP2 membrane accumulation in kidney slices. In vivo, a V2R antagonist caused a severe urinary concentrating defect in rats, which was greatly alleviated by treatment with butaprost. In conclusion, EP2 and EP4 agonists increase AQP2 phosphorylation and trafficking, likely through different signaling pathways. Furthermore, EP2 selective agonists can partially compensate for a nonfunctional V2R, providing a rationale for new treatment strategies for hereditary nephrogenic diabetes insipidus. PMID:21768374

  7. Selective Targeting of the KRAS Codon?12 Mutation Sequence by Pyrrole-Imidazole Polyamide seco-CBI Conjugates.

    PubMed

    Taylor, Rhys D; Chandran, Anandhakumar; Kashiwazaki, Gengo; Hashiya, Kaori; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2015-10-12

    Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon?12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon?12 mutation in a mouse model by using an alkylating hairpin N-methylpyrrole-N-methylimidazole polyamide seco-1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one conjugate (conjugate 4) designed to target the KRAS codon?12 mutation sequence. Herein, we have compared the alkylating activity of 4 against three other conjugates that were also designed to target the KRAS codon?12 mutation sequence. Conjugate 4 displayed greater affinity for the G12D mutation sequence than for the G12V sequence. A computer-minimized model suggested that conjugate 4 could bind more efficiently to the G12D match sequence than to a one-base-pair mismatch sequence. Conjugate 4 was modified for next-generation sequencing. Bind-n-Seq analysis supported the evidence showing that conjugate 4 could target the G12D mutation sequence with exceptionally high affinity and the G12V mutation sequence with much higher affinity than that for the wild-type sequence. PMID:26306751

  8. G? recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets.

    PubMed

    Kaishima, Misato; Ishii, Jun; Fukuda, Nobuo; Kondo, Akihiko

    2015-01-01

    Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a G? recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein ? subunit (G?cyto) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the G? recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity. PMID:26581329

  9. G? recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets

    PubMed Central

    Kaishima, Misato; Ishii, Jun; Fukuda, Nobuo; Kondo, Akihiko

    2015-01-01

    Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a G? recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein ? subunit (G?cyto) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the G? recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity. PMID:26581329

  10. Selective pathogen targeting and macrophage evading carbon nanotubes through dextran sulfate coating and PEGylation for photothermal theranostics.

    PubMed

    Kotagiri, Nalinikanth; Lee, Ju Seok; Kim, Jin-Woo

    2013-06-01

    Single-walled carbon nanotubes (SWNTs) have shown promise as in vivo contrast nanoagents for medical theranostics, in particular photoacoustic and photothermal imaging and therapy, as well as targeted drug delivery systems. However, SWNTs have not proved able to evade biological obstacles, such as opsonization and phagocytosis by macrophage and nonspecific attachments to cells and other biological components in the bloodstream, before reaching target tissues and cells in vivo. Here, we demonstrate the stealth character of dextran sulfate (DS) coated SWNTs (DS-SWNTs) towards human macrophages and other biological barriers using Staphylococcus aureus, a bacterial pathogen, as a model. DS-SWNTs were compared to PEGylated SWNTs, a commonly accepted standard for rendering nanoparticles immune to opsonization. Also a new site-specific conjugation strategy was developed to functionalize antibody (Ab) on DS-SWNT in an upright way, enhancing their targeting efficiency. DS coating was proved to be resistant to opsonins and bacterial cells, demonstrating its potential to provide considerable stealth.character to SWNTs with excellent immunity versus macrophages and other biological barriers, and achieve prolonged blood circulation times. Moreover, the hybrid nanoagents could not only selectively bind to target pathogenic cells upon the controlled Ab attachment but also effectively eradicate pathogens after near-infrared laser irradiation. PMID:23858965

  11. Target Selection for SETI 1. A Catalog of Nearby Habitable Stellar Systems

    E-print Network

    Turnbull, M C; Turnbull, Margaret C.; Tarter, Jill C.

    2002-01-01

    In preparation for the advent of the Allen Telescope Array, the SETI Institute has the need to greatly expand its former list of ~2000 targets compiled for Project Phoenix, a search for extraterrestrial technological signals. In this paper we present a catalog of stellar systems that are potentially habitable to complex life forms (including intelligent life), which comprises the largest portion of the new SETI target list. The Catalog of Nearby Habitable Systems (HabCat) was created from the Hipparcos Catalogue by examining the information on distances, stellar variability, multiplicity, kinematics and spectral classification for the 118,218 stars contained therein. We also make use of information from several other catalogs containing data for Hipparcos stars on X-ray luminosity, CaII H&K activity, rotation, spectral types, kinematics, metallicity, and Stroemgren photometry. Combined with theoretical studies on habitable zones, evolutionary tracks and third body orbital stability, these data were used t...

  12. Targeted Multiplexed Selected Reaction Monitoring Analysis Evaluates Protein Expression Changes of Molecular Risk Factors for Major Psychiatric Disorders

    PubMed Central

    Wesseling, Hendrik; Gottschalk, Michael G.

    2015-01-01

    Background: Extensive research efforts have generated genomic, transcriptomic, proteomic, and functional data hoping to elucidate psychiatric pathophysiology. Selected reaction monitoring, a recently developed targeted proteomic mass spectrometric approach, has made it possible to evaluate previous findings and hypotheses with high sensitivity, reproducibility, and quantitative accuracy. Methods: Here, we have developed a labelled multiplexed selected reaction monitoring assay, comprising 56 proteins previously implicated in the aetiology of major psychiatric disorders, including cell type markers or targets and effectors of known psychopharmacological interventions. We analyzed postmortem anterior prefrontal cortex (Brodmann area 10) tissue of patients diagnosed with schizophrenia (n=22), bipolar disorder (n=23), and major depressive disorder with (n=11) and without (n=11) psychotic features compared with healthy controls (n=22). Results: Results agreed with several previous studies, with the finding of alterations of Wnt-signalling and glutamate receptor abundance predominately in bipolar disorder and abnormalities in energy metabolism across the neuropsychiatric disease spectrum. Calcium signalling was predominantly affected in schizophrenia and affective psychosis. Interestingly, we were able to show a decrease of all 4 tested oligodendrocyte specific proteins (MOG, MBP, MYPR, CNPase) in bipolar disorder and to a lesser extent in schizophrenia and affective psychosis. Finally, we provide new evidence linking ankyrin 3 specifically to affective psychosis and the 22q11.2 deletion syndrome-associated protein septin 5 to schizophrenia. Conclusions: Our study highlights the potential of selected reaction monitoring to evaluate the protein abundance levels of candidate markers of neuropsychiatric spectrum disorders, providing a high throughput multiplex platform for validation of putative disease markers and drug targets. PMID:25539505

  13. Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

    PubMed Central

    Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

    2011-01-01

    Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

  14. 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

  15. Tipping the MYC–MIZ1 balance: targeting the HUWE1 ubiquitin ligase selectively blocks MYC-activated genes

    PubMed Central

    Schaub, Franz X; Cleveland, John L

    2014-01-01

    MYC family oncoproteins (MYC, N-MYC and L-MYC) function as basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors that are activated (i.e., overexpressed) in well over half of all human malignancies (Boxer & Dang, 2001; Beroukhim et al, 2010). In this issue of EMBO Molecular Medicine, Eilers and colleagues (Peter et al, 2014) describe a novel approach to disable MYC, whereby inhibition of the ubiquitin ligase HUWE1 stabilizes MIZ1 and leads to the selective repression of MYC-activated target genes. See also: S Peter et al (December 2014) PMID:25368331

  16. Tipping the MYC-MIZ1 balance: targeting the HUWE1 ubiquitin ligase selectively blocks MYC-activated genes.

    PubMed

    Schaub, Franz X; Cleveland, John L

    2014-12-01

    MYC family oncoproteins (MYC, N?MYC and L?MYC) function as basic helix?loop?helix?leucine zipper (bHLH?Zip) transcription factors that are activated (i.e., overexpressed) in well over half of all human malignancies (Boxer & Dang, 2001; Beroukhim et al, 2010). In this issue of EMBO Molecular Medicine, Eilers and colleagues (Peter et al, 2014) describe a novel approach to disable MYC, whereby inhibition of the ubiquitin ligase HUWE1 stabilizes MIZ1 and leads to the selective repression of MYC?activated target genes. PMID:25368331

  17. Ubiquitin-Dependent And Independent Signals In Selective Autophagy.

    PubMed

    Khaminets, Aliaksandr; Behl, Christian; Dikic, Ivan

    2016-01-01

    Selective autophagy regulates the abundance of specific cellular components via a specialized arsenal of factors, termed autophagy receptors, that target protein complexes, aggregates, and whole organelles into lysosomes. Autophagy receptors bind to LC3/GABARAP proteins on phagophore and autophagosome membranes, and recognize signals on cargoes to deliver them to autophagy. Ubiquitin (Ub), a well-known signal for the degradation of polypeptides in the proteasome, also plays an important role in the recognition of cargoes destined for selective autophagy. In addition, a variety of cargoes are committed to selective autophagy pathways by Ub-independent mechanisms employing protein-protein interaction motifs, Ub-like modifiers, and sugar- or lipid-based signals. In this article we summarize Ub-dependent and independent selective autophagy pathways, and discuss regulatory mechanisms and challenges for future studies. PMID:26437584

  18. Receptor selective ruthenium-somatostatin photosensitizer for cancer targeted photodynamic applications.

    PubMed

    Wang, Tao; Zabarska, Natalia; Wu, Yuzhou; Lamla, Markus; Fischer, Stephan; Monczak, Katharina; Ng, David Y W; Rau, Sven; Weil, Tanja

    2015-08-14

    The efficient conjugation of a ruthenium complex and the peptide hormone somatostatin is presented. The resultant biohybrid offers valuable features for photodynamic therapy such as remarkable cellular selectivity, rapid cell uptake by receptor-mediated endocytosis, efficient generation of (1)O2 upon irradiation, potent phototoxicity as well as low cytotoxicity in the "off"-state. PMID:26153573

  19. Phorbol ester induces elevated oxidative activity and alkalization in a subset of lysosomes

    SciTech Connect

    Chen, Chii-Shiarng )

    2002-01-01

    Background: Lysosomes are acidic organelles that play multiple roles in various cellular oxidative activities such as the oxidative burst during cytotoxic killing. It remains to be determined how lysosomal lumen oxidative activity and pH interact and are regulated. Here, I report the use of fluorescent probes to measure oxidative activity and pH of lysosomes in live macrophages upon treatment with the tumor promotor phorbol 12-myristate 13-acetate (PMA), and provide novel insight regarding the regulation of lysosomal oxidative activity and pH. Results: The substrate used to measure oxidative activity was bovine serum albumin covalently coupled to dihydro-2?, 4,5,6,7,7?-hexafluorofluorescein (OxyBURST Green H2HFF BSA). During pulse-chase procedures with live macrophages, this reduced dye was internalized through an endocytic pathway and accumulated in the lysosomes. Oxidation of this compound results in a dramatic increase of fluorescence intensity. By using low-light level fluorescence microscopy, I determined that phorbol ester treatment results in increased oxidative activity and pH elevation in different subsets of lysosomes. Furthermore, lysosomes with stronger oxidative activity tended to exclude the acidotropic lysosomal indicator, and thus exhibit higher alkalinity. Conclusions: Results indicate that there is a regulatory mechanism between lysosomal oxidative activity and pH. Activation of lysosomal Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase by phorbol ester may result in increase of intralysosomal O2?- and H2O2, concurrent with pH elevation due to consumption of H+ and generation of OH-. Furthermore, effect of phorbol ester on elevated oxidative activity and pH is heterogeneous among total lysosomal population. Higher oxidative activity and/or pH are only observed in subsets of lysosomes.

  20. Selective inhibition of regulatory T cells by targeting PI3K-Akt pathway

    PubMed Central

    Abu-Eid, R; Samara, RN; Ozbun, L; Abdalla, MY; Berzofsky, JA; Friedman, KM; Mkrtichyan, M; Khleif, SN

    2014-01-01

    Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses still are 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, T regulatory cells (Treg). Although the depletion of Treg 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 Treg 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 Treg in comparison to Tconv 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 Treg both in 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 Treg. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg-depletion. PMID:25080445

  1. Mapping Features of HIV-1 Integrase Near Selected Sites on Viral and Target DNA Molecules in an Active Enzyme-DNA Complex by Photo-Cross-Linking

    E-print Network

    Mapping Features of HIV-1 Integrase Near Selected Sites on Viral and Target DNA Molecules-cross-linking approach to address these issues. Our findings suggest that HIV-1 integrase contacts with conserved

  2. Demonstration of the existence of a second, non-lysosomal glucocerebrosidase that is not deficient in Gaucher disease.

    PubMed

    van Weely, S; Brandsma, M; Strijland, A; Tager, J M; Aerts, J M

    1993-03-24

    In addition to the lysosomal glucocerebrosidase, a distinct beta-glucosidase that is also active towards glucosylceramide could be demonstrated in various human tissues and cell types. Subcellular fractionation analysis revealed that the hitherto undescribed glucocerebrosidase is not located in lysosomes but in compartments with a considerably lower density. The non-lysosomal glucocerebrosidase differed in several respects from lysosomal glucocerebrosidase. The non-lysosomal isoenzyme proved to be tightly membrane-bound, whereas lysosomal glucocerebrosidase is weakly membrane-associated. The pH optimum of the non-lysosomal isoenzyme is less acidic than that of lysosomal glucocerebrosidase. Non-lysosomal glucocerebrosidase, in contrast to the lysosomal isoenzyme, was not inhibited by low concentrations of conduritol B-epoxide, was markedly inhibited by taurocholate, was not stimulated in activity by the lysosomal activator protein saposin C, and was not deficient in patients with Gaucher disease. Non-lysosomal glucocerebrosidase proved to be less sensitive to inhibition by castanospermine or deoxynojirimycin but more sensitive to inhibition by D-gluconolactone than the lysosomal glucocerebrosidase. The physiological function of this second, non-lysosomal, glucocerebrosidase is as yet unknown. PMID:8457606

  3. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

    PubMed Central

    Lu, Yingying; Dong, Shichen; Hao, Baixia; Li, Chang; Zhu, Kaiyuan; Guo, Wenjing; Wang, Qian; Cheung, King-Ho; Wong, Connie WM; Wu, Wu-Tian; Markus, Huss; Yue, Jianbo

    2014-01-01

    Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy. PMID:25483964

  4. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV?

    PubMed Central

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMI?), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMI? were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMI?. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMI? impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMI? contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors. PMID:11739797

  5. Protective role of endogenous gangliosides for lysosomal pathology in a cellular model of synucleinopathies.

    PubMed

    Wei, Jianshe; Fujita, Masayo; Nakai, Masaaki; Waragai, Masaaki; Sekigawa, Akio; Sugama, Shuei; Takenouchi, Takato; Masliah, Eliezer; Hashimoto, Makoto

    2009-05-01

    Gangliosides may be involved in the pathogenesis of Parkinson's disease and related disorders, although the precise mechanisms governing this involvement remain unknown. In this study, we determined whether changes in endogenous ganglioside levels affect lysosomal pathology in a cellular model of synucleinopathy. For this purpose, dementia with Lewy body-linked P123H beta-synuclein (beta-syn) neuroblastoma cells transfected with alpha-synuclein were used as a model system because these cells were characterized as having extensive formation of lysosomal inclusions bodies. Treatment of these cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glycosyl ceramide synthase, resulted in various features of lysosomal pathology, including compromised lysosomal activity, enhanced lysosomal membrane permeabilization, and increased cytotoxicity. Consistent with these findings, expression levels of lysosomal membrane proteins, ATP13A2 and LAMP-2, were significantly decreased, and electron microscopy demonstrated alterations in the lysosomal membrane structures. Furthermore, the accumulation of both P123H beta-syn and alpha-synuclein proteins was significant in PDMP-treated cells because of the suppressive effect of PDMP on the autophagy pathway. Finally, the detrimental effects of PDMP on lysosomal pathology were significantly ameliorated by the addition of gangliosides to the cultured cells. These data suggest that endogenous gangliosides may play protective roles against the lysosomal pathology of synucleinopathies. PMID:19349362

  6. Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling

    E-print Network

    Nicola Fameli; Oluseye A. Ogunbayo; Cornelis van Breemen; A. Mark Evans

    2014-01-28

    We demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular calcium (Ca2+) waves. In pulmonary artery smooth muscle cells (PASMCs) nicotinic acid adenine dinucleotide phosphate (NAADP) may trigger increases in cytoplasmic Ca2+ via L-SR junctions, in a manner that requires initial Ca2+ release from lysosomes and subsequent Ca2+-induced Ca2+ release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be health and disease.

  7. Lysosomal responses in the digestive gland of the freshwater mussel, Dreissena polymorpha, experimentally exposed to cadmium

    SciTech Connect

    Giamberini, Laure . E-mail: giamb@sciences.univ-metz.fr; Cajaraville, Miren P.

    2005-06-01

    In order to examine the possible use of lysosomal response as a biomarker of freshwater quality, structural changes of lysosomes were measured by image analysis in the digestive gland of the zebra mussel, Dreissena polymorpha, exposed in laboratory conditions to cadmium. Mussels were exposed to the metal (10 and 200 {mu}g/L) for 3 weeks and randomly collected after 7 and 21 days. At each treatment day, digestive tissues were excised and {beta}-glucuronidase activity was revealed in cryotome sections. Four stereological parameters were calculated: lysosomal volume density, lysosomal surface density, lysosomal surface to volume ratio, and lysosomal numerical density. The changes observed in this study reflected a general activation of the lysosomal system, including an increase in both the number and the size of lysosomes in the digestive gland cells of mussels exposed to cadmium. The digestive lysosomal response in zebra mussels was related to exposure time and to metal concentration, demonstrating the potential of this biomarker in freshwater biomonitoring.

  8. Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

    PubMed

    Davis, Michael J; Eastman, Alison J; Qiu, Yafeng; Gregorka, Brian; Kozel, Thomas R; Osterholzer, John J; Curtis, Jeffrey L; Swanson, Joel A; Olszewski, Michal A

    2015-03-01

    Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-? abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections. PMID:25637026

  9. A new lysosomal storage disorder resembling Morquio syndrome in sibs.

    PubMed

    Perrin, Laurence; Fenneteau, Odile; Ilharreborde, Brice; Capri, Yline; Gérard, Marion; Quoc, Emmanuel Bui; Passemard, Sandrine; Ghoumid, Jamal; Caillaud, Catherine; Froissart, Roseline; Tabet, Anne-Claude; Lebon, Sophie; El Ghouzzi, Vincent; Mazda, Keyvan; Verloes, Alain

    2012-03-01

    We report two male sibs, born from unrelated French Caribbean parents, presenting with an unclassifiable storage disorder. Pregnancy and delivery were uneventful. Stunted growth was noted during the first year of life. Both children have short stature (below - 4SD) with short trunk, barrel chest, micromelia with rhizomelic shortening, severe kyphoscoliosis, pectus carinatum, short hands and feet with metatarsus adductus, and excessive joint laxity of the small joints. Learning difficulties with borderline intelligence quotient (IQ) were noted in one of them. They had no hepatomegaly, no splenomegaly, and no dysmorphism. Skeletal X-rays survey demonstrated generalized platyspondyly with tongue-like deformity of the anterior part of the vertebral bodies, hypoplasia of the odontoid process, generalized epiphyseal dysplasia and abnormally shaped metaphyses. The acetabular roofs had a trident aspect. Ophthalmologic and cardiac examinations were normal. Spine deformity required surgical correction in one of the patient at age 4 years. Lysosomal enzymes assays including N-acetylgalactosamine-6-sulfate sulfatase and ?-galactosidase were normal, excluding mucopolysaccharidoses type IV A and IV B (Morquio syndrome), respectively. Qualitative analysis found traces of dermatan and chondroitin-sulfates in urine, but quantitative glycosaminoglycan excretion fell within normal limits. They were no vacuolated lymphocytes. Abnormal coarse inclusions were present in eosinophils. Mild Alder anomaly was observed in polymorphonuclears. Granulations were discretely metachromatic with toluidine blue. Those morphological anomalies are in favor of a lysosomal storage disease. No inclusions were found in skin fibroblasts. We hypothesize that these two boys have a distinct autosomal recessive or X-linked lysosomal storage disorder of unknown origin that shares clinical and radiological features with Morquio disease. PMID:22330346

  10. Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

    PubMed Central

    Ribbens, Jameson; Zheng, Wei; Southall, Noel; Hu, Xin; Marugan, Juan J.; Ferrer, Marc; Maegawa, Gustavo H. B.

    2011-01-01

    Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets. PMID:22216298

  11. Selective sigma-1 (sigma1) receptor antagonists: emerging target for the treatment of neuropathic pain.

    PubMed

    Díaz, José Luis; Zamanillo, Daniel; Corbera, Jordi; Baeyens, José Manuel; Maldonado, Rafael; Pericàs, Miquel Angel; Vela, José Miguel; Torrens, Antoni

    2009-09-01

    A large number of therapeutic roles have been proposed for sigma(1) receptors but the involvement of sigma(1) receptor in non-acute pain had not been well explored up to now. sigma(1) receptor knock-out mice became available offering us the possibility to study the role of sigma(1) receptor in nociception, particularly in models where central sensitization processes play a significant role. Given the attractive therapeutic potential, we have developed a chemical program aimed at the discovery of novel and selective sigma(1) ligands. Herein we discuss the rational basis of this approach and report preliminary pharmacological results of several chemical series and aspects of their structure-activity relationship on sigma(1) receptor. Functional data in pain models are presented mainly on one series that provide evidence to consider selective sigma(1) receptor antagonists an innovative and alternative approach for treating neuropathic pain. PMID:20021351

  12. Targeting the Parasite's DNA with Methyltriazenyl Purine Analogs Is a Safe, Selective, and Efficacious Antitrypanosomal Strategy

    PubMed Central

    Wanner, Martin J.; Alkhaldi, Abdulsalam A. M.; Ebiloma, Godwin U.; Barnes, Rebecca L.; Kaiser, Marcel; Brun, Reto; McCulloch, Richard; Koomen, Gerrit-Jan

    2015-01-01

    The human and veterinary disease complex known as African trypanosomiasis continues to inflict significant global morbidity, mortality, and economic hardship. Drug resistance and toxic side effects of old drugs call for novel and unorthodox strategies for new and safe treatment options. We designed methyltriazenyl purine prodrugs to be rapidly and selectively internalized by the parasite, after which they disintegrate into a nontoxic and naturally occurring purine nucleobase, a simple triazene-stabilizing group, and the active toxin: a methyldiazonium cation capable of damaging DNA by alkylation. We identified 2-(3-acetyl-3-methyltriazen-1-yl)-6-hydroxypurine (compound 1) as a new lead compound, which showed submicromolar potency against Trypanosoma brucei, with a selectivity index of >500, and it demonstrated a curative effect in animal models of acute trypanosomiasis. We investigated the mechanism of action of this lead compound and showed that this molecule has significantly higher affinity for parasites over mammalian nucleobase transporters, and it does not show cross-resistance with current first-line drugs. Once selectively accumulated inside the parasite, the prodrug releases a DNA-damaging methyldiazonium cation. We propose that ensuing futile cycles of attempted mismatch repair then lead to G2/M phase arrest and eventually cell death, as evidenced by the reduced efficacy of this purine analog against a mismatch repair-deficient (MSH2?/?) trypanosome cell line. The observed absence of genotoxicity, hepatotoxicity, and cytotoxicity against mammalian cells revitalizes the idea of pursuing parasite-selective DNA alkylators as a safe chemotherapeutic option for the treatment of human and animal trypanosomiasis. PMID:26282430

  13. Targeting the Parasite's DNA with Methyltriazenyl Purine Analogs Is a Safe, Selective, and Efficacious Antitrypanosomal Strategy.

    PubMed

    Rodenko, Boris; Wanner, Martin J; Alkhaldi, Abdulsalam A M; Ebiloma, Godwin U; Barnes, Rebecca L; Kaiser, Marcel; Brun, Reto; McCulloch, Richard; Koomen, Gerrit-Jan; de Koning, Harry P

    2015-11-01

    The human and veterinary disease complex known as African trypanosomiasis continues to inflict significant global morbidity, mortality, and economic hardship. Drug resistance and toxic side effects of old drugs call for novel and unorthodox strategies for new and safe treatment options. We designed methyltriazenyl purine prodrugs to be rapidly and selectively internalized by the parasite, after which they disintegrate into a nontoxic and naturally occurring purine nucleobase, a simple triazene-stabilizing group, and the active toxin: a methyldiazonium cation capable of damaging DNA by alkylation. We identified 2-(3-acetyl-3-methyltriazen-1-yl)-6-hydroxypurine (compound 1) as a new lead compound, which showed submicromolar potency against Trypanosoma brucei, with a selectivity index of >500, and it demonstrated a curative effect in animal models of acute trypanosomiasis. We investigated the mechanism of action of this lead compound and showed that this molecule has significantly higher affinity for parasites over mammalian nucleobase transporters, and it does not show cross-resistance with current first-line drugs. Once selectively accumulated inside the parasite, the prodrug releases a DNA-damaging methyldiazonium cation. We propose that ensuing futile cycles of attempted mismatch repair then lead to G2/M phase arrest and eventually cell death, as evidenced by the reduced efficacy of this purine analog against a mismatch repair-deficient (MSH2(-/-)) trypanosome cell line. The observed absence of genotoxicity, hepatotoxicity, and cytotoxicity against mammalian cells revitalizes the idea of pursuing parasite-selective DNA alkylators as a safe chemotherapeutic option for the treatment of human and animal trypanosomiasis. PMID:26282430

  14. Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

    PubMed Central

    Altenhöfer, Sebastian; Radermacher, Kim A.; Kleikers, Pamela W.M.; Wingler, Kirstin

    2015-01-01

    Abstract Significance: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Recent Advances: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Critical Issues: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. Future Directions: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition. Antioxid. Redox Signal. 23, 406–427. PMID:24383718

  15. Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation.

    PubMed

    Fan, Xuelai; Jin, Wu Yang; Lu, Jie; Wang, Jin; Wang, Yu Tian

    2014-03-01

    Rapid and reversible methods for altering the levels of endogenous proteins are critically important for studying biological systems and developing therapeutics. Here we describe a membrane-permeant targeting peptide-based method that rapidly and reversibly knocks down endogenous proteins through chaperone-mediated autophagy in vitro and in vivo. We demonstrate the specificity, efficacy and generalizability of the method by showing efficient knockdown of various proteins, including death associated protein kinase 1 (160 kDa), scaffolding protein PSD-95 (95 kDa) and ?-synuclein (18 kDa), with their respective targeting peptides in a dose-, time- and lysosomal activity-dependent manner in rat neuronal cultures. Moreover, we show that, when given systemically, the peptide system efficiently knocked down the targeted protein in the brains of intact rats. Our study provides a robust and convenient research tool for manipulating endogenous protein levels and may also lead to the development of protein knockdown-based therapeutics for treating human diseases. PMID:24464042

  16. Target Selection for SETI. I. A Catalog of Nearby Habitable Stellar Systems

    NASA Astrophysics Data System (ADS)

    Turnbull, Margaret C.; Tarter, Jill C.

    2003-03-01

    In preparation for the advent of the Allen Telescope Array, the SETI Institute has the need to greatly expand its former list of ~2000 targets compiled for Project Phoenix, a search for extraterrestrial technological signals. In this paper we present a catalog of stellar systems that are potentially habitable to complex life forms (including intelligent life), which comprises the largest portion of the new SETI target list. The Catalog of Nearby Habitable Systems (HabCat) was created from the Hipparcos Catalogue by examining the information on distances, stellar variability, multiplicity, kinematics, and spectral classification for the 118,218 stars contained therein. We also make use of information from several other catalogs containing data for Hipparcos stars on X-ray luminosity, Ca II H and K activity, rotation, spectral types, kinematics, metallicity, and Strömgren photometry. Combined with theoretical studies on habitable zones, evolutionary tracks, and third-body orbital stability, these data are used to remove unsuitable stars from HabCat, leaving a residue of stars that, to the best of our current knowledge, are potentially habitable hosts for complex life. While this catalog will no doubt need to be modified as we learn more about individual objects, the present analysis results in 17,129 Hipparcos ``habstars'' near the Sun (75% within 140 pc), ~2200 of which are known or suspected to be members of binary or triple star systems.

  17. Target Selection for SETI: 1. A Catalog of Nearby Habitable Stellar Systems

    E-print Network

    Margaret C. Turnbull; Jill C. Tarter

    2002-10-31

    In preparation for the advent of the Allen Telescope Array, the SETI Institute has the need to greatly expand its former list of ~2000 targets compiled for Project Phoenix, a search for extraterrestrial technological signals. In this paper we present a catalog of stellar systems that are potentially habitable to complex life forms (including intelligent life), which comprises the largest portion of the new SETI target list. The Catalog of Nearby Habitable Systems (HabCat) was created from the Hipparcos Catalogue by examining the information on distances, stellar variability, multiplicity, kinematics and spectral classification for the 118,218 stars contained therein. We also make use of information from several other catalogs containing data for Hipparcos stars on X-ray luminosity, CaII H&K activity, rotation, spectral types, kinematics, metallicity, and Stroemgren photometry. Combined with theoretical studies on habitable zones, evolutionary tracks and third body orbital stability, these data were used to remove unsuitable stars from HabCat, leaving a residue of stars that, to the best of our current knowledge, are potentially habitable hosts for complex life. While this Catalog will no doubt need to be modified as we learn more about individual objects, the present analysis results in 17,129 Hipparcos "habstars" near the Sun (75% within 140 pc), ~2200 of which are known or suspected to be members of binary or triple star systems.

  18. NAD(P) biosynthesis enzymes as potential targets for selective drug design.

    PubMed

    Magni, G; Di Stefano, M; Orsomando, G; Raffaelli, N; Ruggieri, S

    2009-01-01

    NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed. PMID:19355893

  19. Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia

    PubMed Central

    Lapalombella, Rosa; Sun, Qingxiang; Williams, Katie; Tangeman, Larissa; Jha, Shruti; Zhong, Yiming; Goettl, Virginia; Mahoney, Emilia; Berglund, Caroline; Gupta, Sneha; Farmer, Alicia; Mani, Rajeswaran; Johnson, Amy J.; Lucas, David; Mo, Xiaokui; Daelemans, Dirk; Sandanayaka, Vincent; Shechter, Sharon; McCauley, Dilara; Shacham, Sharon; Kauffman, Michael

    2012-01-01

    The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of XPO1, thereby inhibiting nuclear export of cargo proteins. The clinical relevance of SINEs was explored in chronic lymphocytic leukemia (CLL), a disease associated with recurrent XPO1 mutations. Evidence is presented that SINEs can restore normal regulation to the majority of the dysregulated pathways in CLL both in vitro and in vivo and induce apoptosis of CLL cells with a favorable therapeutic index, with enhanced killing of genomically high-risk CLL cells that are typically unresponsive to traditional therapies. More importantly, SINE slows disease progression, and improves overall survival in the E?-TCL1-SCID mouse model of CLL with minimal weight loss or other toxicities. Together, these findings demonstrate that XPO1 is a valid target in CLL with minimal effects on normal cells and provide a basis for the development of SINEs in CLL and related hematologic malignancies. PMID:23034282

  20. Selective Targeting of a Disease-Related Conformational Isoform of Macrophage Migration Inhibitory Factor Ameliorates Inflammatory Conditions

    PubMed Central

    Thiele, Michael; Tam, Frederick W. K.; Völkel, Dirk; Douillard, Patrice; Schinagl, Alexander; Kühnel, Harald; Smith, Jennifer; McDaid, John P.; Bhangal, Gurjeet; Yu, Mei-Ching; Pusey, Charles D.; Cook, H. Terence; Kovarik, Josef; Magelky, Erica; Bhan, Atul; Rieger, Manfred; Mudde, Geert C.; Ehrlich, Hartmut; Jilma, Bernd; Tilg, Herbert; Moschen, Alexander; Terhorst, Cox; Scheiflinger, Friedrich

    2015-01-01

    Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy. PMID:26209628

  1. Identification of a Selective G1-Phase Benzimidazolone Inhibitor by a Senescence-Targeted Virtual Screen Using Artificial Neural Networks.

    PubMed

    Bilsland, Alan E; Pugliese, Angelo; Liu, Yu; Revie, John; Burns, Sharon; McCormick, Carol; Cairney, Claire J; Bower, Justin; Drysdale, Martin; Narita, Masashi; Sadaie, Mahito; Keith, W Nicol

    2015-09-01

    Cellular senescence is a barrier to tumorigenesis in normal cells, and tumor cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer therapy. However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning-based in silico screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~2M lead-like compounds. One hundred and forty-seven virtual hits were acquired for validation in growth inhibition and senescence-associated ?-galactosidase assays. Among the found hits, a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced senescence-associated ?-galactosidase activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1, and CDC25C. In addition, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long-term treatments. Preliminary structure-activity and structure clustering analyses are reported, and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor-like profile in normal cells, with different pathways affected in cancer cells. PMID:26476078

  2. Self-assembled pentablock copolymers for selective and sustained gene delivery

    SciTech Connect

    Zhang, Bingqi

    2011-05-15

    The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomes of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.

  3. Detection of peptides, proteins, and drugs that selectively interact with protein targets.

    PubMed

    Serebriiskii, Ilya G; Mitina, Olga; Pugacheva, Elena N; Benevolenskaya, Elizaveta; Kotova, Elena; Toby, Garabet G; Khazak, Vladimir; Kaelin, William G; Chernoff, Jonathan; Golemis, Erica A

    2002-11-01

    Genome sequencing has been completed for multiple organisms, and pilot proteomic analyses reported for yeast and higher eukaryotes. This work has emphasized the facts that proteins are frequently engaged in multiple interactions, and that governance of protein interaction specificity is a primary means of regulating biological systems. In particular, the ability to deconvolute complex protein interaction networks to identify which interactions govern specific signaling pathways requires the generation of biological tools that allow the distinction of critical from noncritical interactions. We report the application of an enhanced Dual Bait two-hybrid system to allow detection and manipulation of highly specific protein-protein interactions. We summarize the use of this system to detect proteins and peptides that target well-defined specific motifs in larger protein structures, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction. PMID:12421766

  4. Selective multifaceted E3 ubiquitin ligases barricade extreme defense: Potential therapeutic targets for neurodegeneration and ageing.

    PubMed

    Upadhyay, Arun; Amanullah, Ayeman; Chhangani, Deepak; Mishra, Ribhav; Mishra, Amit

    2015-11-01

    Efficient and regular performance of Ubiquitin Proteasome System and Autophagy continuously eliminate deleterious accumulation of nonnative protiens. In cellular quality control system, E3 ubiquitin ligases are significant employees for defense mechanism against abnormal toxic proteins. Few findings indicate that lack of functions of E3 ubiquitin ligases can be a causative factor of neurodevelopmental disorders, neurodegeneration, cancer and ageing. However, the detailed molecular pathomechanism implying E3 ubiquitin ligases in cellular functions in multifactorial disease conditions are not well understood. This article systematically represents the unique characteristics, molecular nature, and recent developments in the knowledge of neurobiological functions of few crucial E3 ubiquitin ligases. Here, we review recent literature on the roles of E6-AP, HRD1 and ITCH E3 ubiquitin ligases in the neuro-pathobiological mechanisms, with precise focus on the processes of neurodegeneration, and thereby propose new lines of potential targets for therapeutic interventions. PMID:26247845

  5. Site selection and directional models of deserts used for ERBE validation targets

    NASA Technical Reports Server (NTRS)

    Staylor, W. F.

    1986-01-01

    Broadband shortwave and longwave radiance measurements obtained from the Nimbus 7 Earth Radiation Budget scanner were used to develop reflectance and emittance models for the Sahara, Gibson, and Saudi Deserts. These deserts will serve as in-flight validation targets for the Earth Radiation Budget Experiment being flown on the Earth Radiation Budget Satellite and two National Oceanic and Atmospheric Administration polar satellites. The directional reflectance model derived for the deserts was a function of the sum and product of the cosines of the solar and viewing zenith angles, and thus reciprocity existed between these zenith angles. The emittance model was related by a power law of the cosine of the viewing zenith angle.

  6. Model-specific selection of molecular targets for heart failure gene therapy

    PubMed Central

    Katz, Michael G.; Fargnoli, Anthony S.; Tomasulo, Catherine E.; Pritchette, Louella A.; Bridges, Charles R.

    2013-01-01

    Heart failure (HF) is a complex multifaceted problem of abnormal ventricular function and structure. In recent years, new information has been accumulated allowing for a more detailed understanding of the cellular and molecular alterations that are the underpinnings of diverse causes of HF, including myocardial ischemia, pressure-overload, volume-overload or intrinsic cardiomyopathy. Modern pharmacological approaches to treat HF have had a significant impact on the course of the disease, although they do not reverse the underlying pathological state of the heart. Therefore gene-based therapy holds a great potential as a targeted treatment for cardiovascular diseases. Here, we survey the relative therapeutic efficacy of genetic modulation of ?-adrenergic receptor signaling, Ca2+ handling proteins and angiogenesis in the most common extrinsic models of HF. PMID:21954055

  7. Vesicle Leakage Reflects the Target Selectivity of Antimicrobial Lipopeptides from Bacillus subtilis.

    PubMed

    Fiedler, Sebastian; Heerklotz, Heiko

    2015-11-17

    Cyclic lipopeptides act against a variety of plant pathogens and are thus highly efficient crop-protection agents. Some pesticides contain Bacillus subtilis strains that produce lipopeptide families, such as surfactins (SF), iturins (IT), and fengycins (FE). The antimicrobial activity of these peptides is mainly mediated by permeabilizing cellular membranes. We used a fluorescence-lifetime based leakage assay to examine the effect of individual lipid components in model membranes on lipopeptide activity. Leakage induction by FE was strongly inhibited by cholesterol (CHOL) as well as by phosphatidylethanolamine (PE) and -glycerol (PG) lipids. Already moderate amounts of CHOL increased the tolerable FE content in membranes by an order of magnitude to 0.5 FE per PC + CHOL. This indicates reduced FE-lipid demixing and aggregation, which is known to be required for membrane permeabilization and explains the strong inhibition by CHOL. Ergosterol (ERG) had a weak antagonistic effect. This confirms results of microbiological tests and agrees with the fungicidal activity and selectivity of FE. SF is known to be much less selective in its antimicrobial action. In line with this, liposome leakage by SF was little affected by sterols and PE. Interestingly, PG increased SF activity and changed its leakage mechanism toward all-or-none, suggesting more specific, larger, and/or longer-lived defect structures. This may be because of the reduced energetic cost of locally accumulating anionic SF in an anionic lipid matrix. IT was found largely inactive in our assays. B. subtilis QST713 produces the lipopeptides in a ratio of 6 mol SF: 37 mol FE: 57 mol IT. Leakage induced by this native mixture was inhibited by CHOL and PE, but unaffected by ERG and by PG in the absence of PE. Note that fungi contain anionic lipids, but little PE. Hence, our data explain the strong, fungicidal activity and selectivity of B. subtilis QST713 lipopeptides. PMID:26588567

  8. Unnatural polyketide analogues selectively target the HER signaling pathway in human breast cancer cells.

    PubMed

    Kwon, Seok Joon; Kim, Moon Il; Ku, Bosung; Coulombel, Lydie; Kim, Jin-Hwan; Shawky, Joseph H; Linhardt, Robert J; Dordick, Jonathan S

    2010-03-01

    Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets. Several plant polyphenols including the type III polyketide synthase products (genistein, curcumin, resveratrol, and epigallocatechin-3-galate) possess chemopreventive activity, primarily as a result of RTK inhibition. However, only a small fraction of the polyphenolic structural universe has been evaluated. Along these lines, we have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogues with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme A (CoA) and methylmalonyl-CoA as extender substrates. The resulting polyketide analogues possessed a similar structural polyketide backbone (aromatic-2-pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT-474 cells resulted in identification of several trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of nontransformed MCF-10A breast cells (IC(50)>100 microM). Specifically, 4-trifluoromethylcinnamoyl pyrone (compound 2 e) was highly potent (IC(50)<200 nM) among the test compounds toward proliferation of several breast cancer cell lines. This breadth of activity likely stems from the ability of compound 2 e to inhibit the phosphorylation of HER1, HER2, and HER3. Therefore, these polyketide analogues might prove to be useful drug candidates for potential breast cancer therapy. PMID:20058253

  9. Unnatural Polyketide Analogues Selectively Target the HER Signaling Pathway in Human Breast Cancer Cells

    PubMed Central

    Kwon, Seok Joon; Kim, Moon Il; Ku, Bosung; Coulombel, Lydie; Kim, Jin-Hwan; Shawky, Joseph H.; Linhardt, Robert J.; Dordick, Jonathan S.

    2010-01-01

    Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets. Several plant polyphenols including the type III polyketide synthase products (genistein, curcumin, resveratrol, and epigallocatechin-3-galate) possess chemopreventive activity, primarily as a result of RTK inhibition. However, only a small fraction of the polyphenolic structural universe has been evaluated. Along these lines, we have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogs with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme A (CoA) and methylmalonyl-CoA as extender substrates. The resulting polyketide analogs possessed a similar strucutral polyketide backbone (aromatic-2-pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT-474 cells resulted in identification of several trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of nontransformed MCF-10A breast cells (IC50 > 100 µm). Specifically, 4-trifluoromethylcinnamoyl pyrone (compound 2e) was highly potent (IC50 < 200 nm) among the test compounds toward proliferation of several breast cancer cell lines. This breadth of activity likely stems from the ability of compound 2e to inhibit the phosphorylation of HER1, HER2, and HER3. Therefore, these polyketide analogs might prove to be useful drug candidates for potential breast cancer therapy. PMID:20058253

  10. Antibodies to selected minor target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA).

    PubMed

    Talor, M V; Stone, J H; Stebbing, J; Barin, J; Rose, N R; Burek, C L

    2007-10-01

    In patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C-ANCA) and perinuclear (P-ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C-ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P-ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti-PR3 and anti-MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well-defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF-positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF-negative (-) sera (P < 0.01). Patients with IF(+) PR3(-)MPO(-) sera showed the most varied reactivity to the minor antigens. Among the IF(+) groups, the IF(+) PR3(+)/MPO(-) sera showed the lowest reactivity to the minor antigens. Patients with well-defined ANCA specificities, e.g. the PR3-ANCA response associated with Wegener's granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes. PMID:17614969

  11. Targeting ferritin receptors for the selective delivery of imaging and therapeutic agents to breast cancer cells

    NASA Astrophysics Data System (ADS)

    Geninatti Crich, S.; Cadenazzi, M.; Lanzardo, S.; Conti, L.; Ruiu, R.; Alberti, D.; Cavallo, F.; Cutrin, J. C.; Aime, S.

    2015-04-01

    In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 ?g ml-1 (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation.In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 ?g ml-1 (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation. Electronic supplementary information (ESI) available: Competition studies with free apoferritin, Fig. S1; APO-FITC intracellular distribution by immunofluorescence, Fig. S2. See DOI: 10.1039/c5nr00352k

  12. Targeting ferritin receptors for the selective delivery of imaging and therapeutic agents to breast cancer cells.

    PubMed

    Geninatti Crich, S; Cadenazzi, M; Lanzardo, S; Conti, L; Ruiu, R; Alberti, D; Cavallo, F; Cutrin, J C; Aime, S

    2015-04-21

    In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 ?g ml(-1) (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation. PMID:25786779

  13. Gene therapy for the neurological manifestations in lysosomal storage disorders.

    PubMed

    Cheng, Seng H

    2014-09-01

    Over the past several years, considerable progress has been made in the development of gene therapy as a therapeutic strategy for a variety of inherited metabolic diseases, including neuropathic lysosomal storage disorders (LSDs). The premise of gene therapy for this group of diseases is borne of findings that genetic modification of a subset of cells can provide a more global benefit by virtue of the ability of the secreted lysosomal enzymes to effect cross-correction of adjacent and distal cells. Preclinical studies in small and large animal models of these disorders support the application of either a direct in vivo approach using recombinant adeno-associated viral vectors or an ex vivo strategy using lentiviral vector-modified hematopoietic stem cells to correct the neurological component of these diseases. Early clinical studies utilizing both approaches have begun or are in late-stage planning for a small number of neuropathic LSDs. Although initial indications from these studies are encouraging, it is evident that second-generation vectors that exhibit a greater safety profile and transduction activity may be required before this optimism can be fully realized. Here, I review recent progress and the remaining challenges to treat the neurological aspects of various LSDs using this therapeutic paradigm. PMID:24683200

  14. Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

    PubMed

    Cano, Victoria; March, Catalina; Insua, Jose Luis; Aguiló, Nacho; Llobet, Enrique; Moranta, David; Regueiro, Verónica; Brennan, Gerard P; Millán-Lou, Maria Isabel; Martín, Carlos; Garmendia, Junkal; Bengoechea, José A

    2015-11-01

    Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K.?pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K.?pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K.?pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K.?pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K.?pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis. PMID:26045209

  15. Gene therapy for the neurological manifestations in lysosomal storage disorders

    PubMed Central

    Cheng, Seng H.

    2014-01-01

    Over the past several years, considerable progress has been made in the development of gene therapy as a therapeutic strategy for a variety of inherited metabolic diseases, including neuropathic lysosomal storage disorders (LSDs). The premise of gene therapy for this group of diseases is borne of findings that genetic modification of a subset of cells can provide a more global benefit by virtue of the ability of the secreted lysosomal enzymes to effect cross-correction of adjacent and distal cells. Preclinical studies in small and large animal models of these disorders support the application of either a direct in vivo approach using recombinant adeno-associated viral vectors or an ex vivo strategy using lentiviral vector-modified hematopoietic stem cells to correct the neurological component of these diseases. Early clinical studies utilizing both approaches have begun or are in late-stage planning for a small number of neuropathic LSDs. Although initial indications from these studies are encouraging, it is evident that second-generation vectors that exhibit a greater safety profile and transduction activity may be required before this optimism can be fully realized. Here, I review recent progress and the remaining challenges to treat the neurological aspects of various LSDs using this therapeutic paradigm. PMID:24683200

  16. Metallothioneins as dynamic markers for brain disease in lysosomal disorders

    PubMed Central

    Cesani, Martina; Cavalca, Eleonora; Macco, Romina; Leoncini, Giuseppe; Terreni, Maria Rosa; Lorioli, Laura; Furlan, Roberto; Comi, Giancarlo; Doglioni, Claudio; Zacchetti, Daniele; Sessa, Maria; Scherzer, Clemens R; Biffi, Alessandra

    2014-01-01

    Objective To facilitate development of novel disease-modifying therapies for lysosomal storage disorder (LSDs) characterized by nervous system involvement such as metachromatic leukodystrophy (MLD), molecular markers for monitoring disease progression and therapeutic response are needed. To this end, we sought to identify blood transcripts associated with the progression of MLD. Methods Genome-wide expression analysis was performed in primary T lymphocytes of 24 patients with MLD compared to 24 age- and sex-matched healthy controls. Genes associated with MLD were identified, confirmed on a quantitative polymerase chain reaction platform, and replicated in an independent patient cohort. mRNA and protein expression of the prioritized gene family of metallothioneins was evaluated in postmortem patient brains and in mouse models representing 6 other LSDs. Metallothionein expression during disease progression and in response to specific treatment was evaluated in 1 of the tested LSD mouse models. Finally, a set of in vitro studies was planned to dissect the biological functions exerted by this class of molecules. Results Metallothionein genes were significantly overexpressed in T lymphocytes and brain of patients with MLD and generally marked nervous tissue damage in the LSDs here evaluated. Overexpression of metallothioneins correlated with measures of disease progression in mice and patients, whereas their levels decreased in mice upon therapeutic treatment. In vitro studies indicated that metallothionein expression is regulated in response to oxidative stress and inflammation, which are biochemical hallmarks of lysosomal storage diseases. Interpretation Metallothioneins are potential markers of neurologic disease processes and treatment response in LSDs. PMID:24242821

  17. Predicting the Interactome of Xanthomonas oryzae pathovar oryzae for target selection and DB service

    PubMed Central

    Kim, Jeong-Gu; Park, Daeui; Kim, Byoung-Chul; Cho, Seong-Woong; Kim, Yeong Tae; Park, Young-Jin; Cho, Hee Jung; Park, Hyunseok; Kim, Ki-Bong; Yoon, Kyong-Oh; Park, Soo-Jun; Lee, Byoung-Moo; Bhak, Jong

    2008-01-01

    Background Protein-protein interactions (PPIs) play key roles in various cellular functions. In addition, some critical inter-species interactions such as host-pathogen interactions and pathogenicity occur through PPIs. Phytopathogenic bacteria infect hosts through attachment to host tissue, enzyme secretion, exopolysaccharides production, toxins release, iron acquisition, and effector proteins secretion. Many such mechanisms involve some kind of protein-protein interaction in hosts. Our first aim was to predict the whole protein interaction pairs (interactome) of Xanthomonas oryzae pathovar oryzae (Xoo) that is an important pathogenic bacterium that causes bacterial blight (BB) in rice. We developed a detection protocol to find possibly interacting proteins in its host using whole genome PPI prediction algorithms. The second aim was to build a DB server and a bioinformatic procedure for finding target proteins in Xoo for developing pesticides that block host-pathogen protein interactions within critical biochemical pathways. Description A PPI network in Xoo proteome was predicted by bioinformatics algorithms: PSIMAP, PEIMAP, and iPfam. We present the resultant species specific interaction network and host-pathogen interaction, XooNET. It is a comprehensive predicted initial PPI data for Xoo. XooNET can be used by experimentalists to pick up protein targets for blocking pathological interactions. XooNET uses most of the major types of PPI algorithms. They are: 1) Protein Structural Interactome MAP (PSIMAP), a method using structural domain of SCOP, 2) Protein Experimental Interactome MAP (PEIMAP), a common method using public resources of experimental protein interaction information such as HPRD, BIND, DIP, MINT, IntAct, and BioGrid, and 3) Domain-domain interactions, a method using Pfam domains such as iPfam. Additionally, XooNET provides information on network properties of the Xoo interactome. Conclusion XooNET is an open and free public database server for protein interaction information for Xoo. It contains 4,538 proteins and 26,932 possible interactions consisting of 18,503 (PSIMAP), 3,118 (PEIMAP), and 8,938 (iPfam) pairs. In addition, XooNET provides 3,407 possible interaction pairs between two sets of proteins; 141 Xoo proteins that are predicted as membrane proteins and rice proteomes. The resultant interacting partners of a query protein can be easily retrieved by users as well as the interaction networks in graphical web interfaces. XooNET is freely available from . PMID:18215330

  18. Modulating carnitine levels by targeting its biosynthesis pathway – selective inhibition of ?-butyrobetaine hydroxylase

    PubMed Central

    Rydzik, Anna M.; Chowdhury, Rasheduzzaman; Kochan, Grazyna T.; Williams, Sophie T.; McDonough, Michael A.; Kawamura, Akane; Schofield, Christopher J.

    2015-01-01

    Carnitine is essential for fatty acid metabolism, but is associated with both health benefits and risks, especially heart diseases. We report the identification of potent, selective and cell active inhibitors of ?-butyrobetaine hydroxylase (BBOX), which catalyses the final step of carnitine biosynthesis in animals. A crystal structure of BBOX in complex with a lead inhibitor reveals that it binds in two modes, one of which adopts an unusual ‘U-shape’ conformation stabilised by inter- and intra-molecular ?-stacking interactions. Conformational changes observed on binding of the inhibitor to BBOX likely reflect those occurring in catalysis; they also rationalise the inhibition of BBOX by high levels of its substrate ?-butyrobetaine (GBB), as observed both with isolated BBOX protein and in cellular studies.

  19. Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

    PubMed Central

    Sok, Devin; van Gils, Marit J.; Pauthner, Matthias; Julien, Jean-Philippe; Saye-Francisco, Karen L.; Hsueh, Jessica; Briney, Bryan; Lee, Jeong Hyun; Le, Khoa M.; Lee, Peter S.; Hua, Yuanzi; Seaman, Michael S.; Moore, John P.; Ward, Andrew B.; Wilson, Ian A.; Sanders, Rogier W.; Burton, Dennis R.

    2014-01-01

    Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC50 of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family. PMID:25422458

  20. The Rapamycin-Binding Domain Governs Substrate Selectivity by the Mammalian Target of Rapamycin

    PubMed Central

    McMahon, Lloyd P.; Choi, Kin M.; Lin, Tai-An; Abraham, Robert T.; Lawrence, John C.

    2002-01-01

    The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70S6K. Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70S6K, were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells. PMID:12370290

  1. Hyperthermia Selectively Targets Human Papillomavirus in Cervical Tumors via p53-Dependent Apoptosis.

    PubMed

    Oei, Arlene L; van Leeuwen, Caspar M; Ten Cate, Rosemarie; Rodermond, Hans M; Buist, Marrije R; Stalpers, Lukas J A; Crezee, Johannes; Kok, H Petra; Medema, Jan Paul; Franken, Nicolaas A P

    2015-12-01

    Human papillomavirus (HPV) is associated with cervical cancer, the third most common cancer in women. The high-risk HPV types 16 and 18 are found in over 70% of cervical cancers and produce the oncoprotein, early protein 6 (E6), which binds to p53 and mediates its ubiquitination and degradation. Targeting E6 has been shown to be a promising treatment option to eliminate HPV-positive tumor cells. In addition, combined hyperthermia with radiation is a very effective treatment strategy for cervical cancer. In this study, we examined the effect of hyperthermia on HPV-positive cells using cervical cancer cell lines infected with HPV 16 and 18, in vivo tumor models, and ex vivo-treated patient biopsies. Strikingly, we demonstrate that a clinically relevant hyperthermia temperature of 42°C for 1 hour resulted in E6 degradation, thereby preventing the formation of the E6-p53 complex and enabling p53-dependent apoptosis and G2-phase arrest. Moreover, hyperthermia combined with p53 depletion restored both the cell-cycle distribution and apoptosis to control levels. Collectively, our findings provide new insights into the treatment of HPV-positive cervical cancer and suggest that hyperthermia therapy could improve patient outcomes. Cancer Res; 75(23); 5120-9. ©2015 AACR. PMID:26573798

  2. Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-­lysosome fusion

    E-print Network

    Brandstaetter, Hemma; Kishi-­Itakura, Chieko; Tumbarello, David A.; Manstein, Dietmar J.; Buss, Folma

    2015-01-28

    major selective impact on the autophagy pathway. Introduction Autophagy is a cellular degradation pathway to maintain cel- lular homeostasis in times of starvation and to eliminate long- lived or misfolded proteins, damaged organelles as well as cyto... in the morphology of late endo- cytic organelles in the form of enlarged swollen lysosomes following loss of MYO1C.16 Here, we demonstrate that changes in cellular cholesterol trafficking in MYO1C-depleted cells cause a significant increase in total intracel- lular...

  3. Membrane Vesicles Shed by Legionella pneumophila Inhibit Fusion of Phagosomes with Lysosomes

    PubMed Central

    Fernandez-Moreira, Esteban; Helbig, Juergen H.; Swanson, Michele S.

    2006-01-01

    When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharide-specific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only ?25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas ?75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered ?72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes. PMID:16714556

  4. Loss of glucocerebrosidase 1 activity causes lysosomal dysfunction and ?-synuclein aggregation

    PubMed Central

    Bae, Eun-Jin; Yang, Na Young; Lee, Cheolsoon; Lee, He-Jin; Kim, Seokjoong; Sardi, Sergio Pablo; Lee, Seung-Jae

    2015-01-01

    Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes ?-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When ?-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of ?-synuclein aggregates. PMID:25813221

  5. The Endosymbiotic Bacterium Wolbachia Selectively Kills Male Hosts by Targeting the Masculinizing Gene.

    PubMed

    Fukui, Takahiro; Kawamoto, Munetaka; Shoji, Keisuke; Kiuchi, Takashi; Sugano, Sumio; Shimada, Toru; Suzuki, Yutaka; Katsuma, Susumu

    2015-07-01

    Pathogens are known to manipulate the reproduction and development of their hosts for their own benefit. Wolbachia is an endosymbiotic bacterium that infects a wide range of insect species. Wolbachia is known as an example of a parasite that manipulates the sex of its host's progeny. Infection of Ostrinia moths by Wolbachia causes the production of all-female progeny, however, the mechanism of how Wolbachia accomplishes this male-specific killing is unknown. Here we show for the first time that Wolbachia targets the host masculinizing gene of Ostrinia to accomplish male-killing. We found that Wolbachia-infected O. furnacalis embryos do not express the male-specific splice variant of doublesex, a gene which acts at the downstream end of the sex differentiation cascade, throughout embryonic development. Transcriptome analysis revealed that Wolbachia infection markedly reduces the mRNA level of Masc, a gene that encodes a protein required for both masculinization and dosage compensation in the silkworm Bombyx mori. Detailed bioinformatic analysis also elucidated that dosage compensation of Z-linked genes fails in Wolbachia-infected O. furnacalis embryos, a phenomenon that is extremely similar to that observed in Masc mRNA-depleted male embryos of B. mori. Finally, injection of in vitro transcribed Masc cRNA into Wolbachia-infected embryos rescued male progeny. Our results show that Wolbachia-induced male-killing is caused by a failure of dosage compensation via repression of the host masculinizing gene. Our study also shows a novel strategy by which a pathogen hijacks the host sex determination cascade. PMID:26172536

  6. Targeted selective treatment for worm management--how do we sell rational programs to farmers?

    PubMed

    van Wyk, J A; Hoste, H; Kaplan, R M; Besier, R B

    2006-07-31

    Seriously escalating global anthelmintic resistance in gastrointestinal nematodes of small ruminants has spawned a variety of alternatives to anthelmintics for worm management, based on the need for sustainable Integrated Parasite Management (sIPM). Pivotal to the sIPM approach is the concept of refugia, the proportion of a given parasite population that escapes exposure to control measures. By balancing drug applications with the maintenance of refugia, the accumulation of anthelmintic resistance alleles in worm populations can be considerably delayed, while still providing good levels of control. The over-dispersed nature of parasitic infections provides an opportunity to achieve this balance, by targeting treatments to the members of a flock or herd that are least tolerant to nematode infection. However, implementation of this strategy has only recently become feasible, with the development of the FAMACHA((c)) system for clinical evaluation of anaemia due to haemonchosis. Subsequently, the use of milk yields has proven an effective indicator in dairy goats infected predominantly with nematodes other than Haemonchus contortus. In addition, short-term weight changes and perhaps also body condition scoring may provide indices of parasitism, permitting the rapid identification of animals likely to benefit from treatment. However, sIPM and refugia-based approaches are more complex than whole-flock treatments in conventional programs, and adoption by farmers is most likely where the theoretical basis is understood. As close communication with informed advisors is generally limited, there is a danger that sIPM will remain a theoretical concept without alternative modes of communication. The development of computer-based decision support programs, which use epidemiological, seasonal and clinical information to provide recommendations for specific situations, should be accorded high priority in the future development of worm management systems. PMID:16774807

  7. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance

    PubMed Central

    Fedorov, Oleg; Castex, Josefina; Tallant, Cynthia; Owen, Dafydd R.; Martin, Sarah; Aldeghi, Matteo; Monteiro, Octovia; Filippakopoulos, Panagis; Picaud, Sarah; Trzupek, John D.; Gerstenberger, Brian S.; Bountra, Chas; Willmann, Dominica; Wells, Christopher; Philpott, Martin; Rogers, Catherine; Biggin, Philip C.; Brennan, Paul E.; Bunnage, Mark E.; Schüle, Roland; Günther, Thomas; Knapp, Stefan; Müller, Susanne

    2015-01-01

    Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5?-triphosphate)–driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine–dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes. PMID:26702435

  8. Novel ROS-activated agents utilize a tethered amine to selectively target acute myeloid leukemia

    PubMed Central

    Bell-Horwath, Tiffany R.; Vadukoot, Anish Kizhakkekkara; Thowfeik, Fathima Shazna; Li, Guorui; Wunderlich, Mark; Mulloy, James C.; Merino, Edward J.

    2013-01-01

    This study explores the possible use of reactive oxygen-activated DNA modifying agents against acute myeloid leukemia (AML). A key amine on the lead agent was investigated via cytotoxicity assays and was found necessary for potency. The two best compounds were screened via the NCI-60 cell panel. These two compounds had potency between 200 and 800 nM against many of the leukemia cancer cell types. Subsequent experiments explored activity against a transformed AML model that mimics the molecular signatures identified in primary AML patient samples. A lead compound had an IC50 of 760 nM against this AML cell line as well as a therapeutic index of 7.7 ± 3 between the transformed AML model cell line and non-cancerous human CD34+ blood stem/progenitor cells (UCB). The selectivity was much greater than the mainstays of AML treatment: doxorubicin and cytarabine. This manuscript demonstrates that this novel type of agent may be useful against AML. PMID:23578690

  9. Prey pursuit strategy of Japanese horseshoe bats during an in-flight target-selection task.

    PubMed

    Kinoshita, Yuki; Ogata, Daiki; Watanabe, Yoshiaki; Riquimaroux, Hiroshi; Ohta, Tetsuo; Hiryu, Shizuko

    2014-09-01

    The prey pursuit behavior of Japanese horseshoe bats (Rhinolophus ferrumequinum nippon) was investigated by tasking bats during flight with choosing between two tethered fluttering moths. Echolocation pulses were recorded using a telemetry microphone mounted on the bat combined with a 17-channel horizontal microphone array to measure pulse directions. Flight paths of the bat and moths were monitored using two high-speed video cameras. Acoustical measurements of returning echoes from fluttering moths were first collected using an ultrasonic loudspeaker, turning the head direction of the moth relative to the loudspeaker from 0° (front) to 180° (back) in the horizontal plane. The amount of acoustical glints caused by moth fluttering varied with the sound direction, reaching a maximum at 70°-100° in the horizontal plane. In the flight experiment, moths chosen by the bat fluttered within or moved across these angles relative to the bat's pulse direction, which would cause maximum dynamic changes in the frequency and amplitude of acoustical glints during flight. These results suggest that echoes with acoustical glints containing the strongest frequency and amplitude modulations appear to attract bats for prey selection. PMID:24958227

  10. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance.

    PubMed

    Fedorov, Oleg; Castex, Josefina; Tallant, Cynthia; Owen, Dafydd R; Martin, Sarah; Aldeghi, Matteo; Monteiro, Octovia; Filippakopoulos, Panagis; Picaud, Sarah; Trzupek, John D; Gerstenberger, Brian S; Bountra, Chas; Willmann, Dominica; Wells, Christopher; Philpott, Martin; Rogers, Catherine; Biggin, Philip C; Brennan, Paul E; Bunnage, Mark E; Schüle, Roland; Günther, Thomas; Knapp, Stefan; Müller, Susanne

    2015-11-01

    Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes. PMID:26702435

  11. Microcystins Alter Chemotactic Behavior in Caenorhabditis elegans by Selectively Targeting the AWA Sensory Neuron

    PubMed Central

    Moore, Caroline E.; Lein, Pamela J.; Puschner, Birgit

    2014-01-01

    Harmful algal blooms expose humans and animals to microcystins (MCs) through contaminated drinking water. While hepatotoxicity following acute exposure to MCs is well documented, neurotoxicity after sub-lethal exposure is poorly understood. We developed a novel statistical approach using a generalized linear model and the quasibinomial family to analyze neurotoxic effects in adult Caenorhabditis elegans exposed to MC-LR or MC-LF for 24 h. Selective effects of toxin exposure on AWA versus AWC sensory neuron function were determined using a chemotaxis assay. With a non-monotonic response MCs altered AWA but not AWC function, and MC-LF was more potent than MC-LR. To probe a potential role for protein phosphatases (PPs) in MC neurotoxicity, we evaluated the chemotactic response in worms exposed to the PP1 inhibitor tautomycin or the PP2A inhibitor okadaic acid for 24 h. Okadaic acid impaired both AWA and AWC function, while tautomycin had no effect on function of either neuronal cell type at the concentrations tested. These findings suggest that MCs alter the AWA neuron at concentrations that do not cause AWC toxicity via mechanisms other than PP inhibition. PMID:24918360

  12. HIV-1 Pre-Integration Complexes Selectively Target Decondensed Chromatin in the Nuclear Periphery

    PubMed Central

    Albanese, Alberto; Arosio, Daniele; Terreni, Mariaelena; Cereseto, Anna

    2008-01-01

    Integration of the double-stranded DNA copy of the HIV-1 genome into host chromosomal DNA is a requirement for efficient viral replication. Integration preferentially occurs within active transcription units, however chromosomal site specificity does not correlate with any strong primary sequence. To investigate whether the nuclear architecture may affect viral integration we have developed an experimental system where HIV-1 viral particles can be visualized within the nuclear compartment. Fluorescently labeled HIV-1 virions were engineered by fusing integrase, the viral protein that catalyzes the integration reaction, to fluorescent proteins. Viral tests demonstrate that the infectivity of fluorescent virions, including the integration step, is not altered as compared to wild-type virus. 3-D confocal microscopy allowed a detailed analysis of the spatial and temporal distribution of the pre-integration complexes (PICs) within the nucleus at different moments following infection; the fluorescently labeled PICs preferentially distribute in decondensed areas of the chromatin with a striking positioning in the nuclear periphery, while heterochromatin regions are largely disfavored. These observations provide a first indication of how the nuclear architecture may initially orient the selection of retroviral integration sites. PMID:18545681

  13. Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization

    E-print Network

    Sargeant, Timothy J.; Lloyd-Lewis, Bethan; Resemann, Henrike K.; Ramos-Montoya, Antonio; Skepper, Jeremy; Watson, Christine J.

    2014-10-05

    We have previously demonstrated that Stat3 regulates lysosomal mediated-programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death...

  14. Mechanism of IS200/IS605 Family DNA Transposases: Activation and Transposon-Directed Target Site Selection

    PubMed Central

    Barabas, Orsolya; Ronning, Donald R.; Guynet, Catherine; Hickman, Alison Burgess; Ton-Hoang, Bao; Chandler, Michael; Dyda, Fred

    2009-01-01

    SUMMARY The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and allow us to propose a molecular model for the entire transposition reaction. PMID:18243097

  15. Mice doubly-deficient in lysosomal hexosaminidase A and neuraminidase 4 show epileptic crises and rapid neuronal loss.

    PubMed

    Seyrantepe, Volkan; Lema, Pablo; Caqueret, Aurore; Dridi, Larbi; Bel Hadj, Samar; Carpentier, Stephane; Boucher, Francine; Levade, Thierry; Carmant, Lionel; Gravel, Roy A; Hamel, Edith; Vachon, Pascal; Di Cristo, Graziella; Michaud, Jacques L; Morales, Carlos R; Pshezhetsky, Alexey V

    2010-09-01

    Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the ?-subunit of lysosomal ?-hexosaminidase A, which converts G(M2) to G(M3) ganglioside. Hexa(-/-) mice, depleted of ?-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G(M2) ganglioside via a lysosomal sialidase into glycolipid G(A2), which is further processed by ?-hexosaminidase B to lactosyl-ceramide, thereby bypassing the ?-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4(-/-);Hexa(-/-)) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa(-/-) or Neu4(-/-) siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating G(M2) ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa(-/-) mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa(-/-) mice. PMID:20862357

  16. Selective Targeting of CTNNB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs

    PubMed Central

    Uitdehaag, Joost C. M.; de Roos, Jeroen A. D. M.; van Doornmalen, Antoon M.; Prinsen, Martine B. W.; Spijkers-Hagelstein, Jill A. P.; de Vetter, Judith R. F.; de Man, Jos; Buijsman, Rogier C.; Zaman, Guido J. R.

    2015-01-01

    The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for ?-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes. PMID:26018524

  17. Mixed methods evaluation of targeted selective anthelmintic treatment by resource-poor smallholder goat farmers in Botswana.

    PubMed

    Walker, Josephine G; Ofithile, Mphoeng; Tavolaro, F Marina; van Wyk, Jan A; Evans, Kate; Morgan, Eric R

    2015-11-30

    Due to the threat of anthelmintic resistance, livestock farmers worldwide are encouraged to selectively apply treatments against gastrointestinal nematodes (GINs). Targeted selective treatment (TST) of individual animals would be especially useful for smallholder farmers in low-income economies, where cost-effective and sustainable intervention strategies will improve livestock productivity and food security. Supporting research has focused mainly on refining technical indicators for treatment, and much less on factors influencing uptake and effectiveness. We used a mixed method approach, whereby qualitative and quantitative approaches are combined, to develop, implement and validate a TST system for GINs in small ruminants, most commonly goats, among smallholder farmers in the Makgadikgadi Pans region of Botswana, and to seek better understanding of system performance within a cultural context. After the first six months of the study, 42 out of 47 enrolled farmers were followed up; 52% had monitored their animals using the taught inspection criteria and 26% applied TST during this phase. Uptake level showed little correlation with farmer characteristics, such as literacy and size of farm. Herd health significantly improved in those herds where anthelmintic treatment was applied: anaemia, as assessed using the five-point FAMACHA(©) scale, was 0.44-0.69 points better (95% confidence interval) and body condition score was 0.18-0.36 points better (95% C.I., five-point scale) in treated compared with untreated herds. Only targeting individuals in greatest need led to similar health improvements compared to treating the entire herd, leading to dose savings ranging from 36% to 97%. This study demonstrates that TST against nematodes can be implemented effectively by resource-poor farmers using a community-led approach. The use of mixed methods provides a promising system to integrate technical and social aspects of TST programmes for maximum uptake and effect. PMID:26493540

  18. Mixed methods evaluation of targeted selective anthelmintic treatment by resource-poor smallholder goat farmers in Botswana

    PubMed Central

    Walker, Josephine G.; Ofithile, Mphoeng; Tavolaro, F. Marina; van Wyk, Jan A.; Evans, Kate; Morgan, Eric R.

    2015-01-01

    Due to the threat of anthelmintic resistance, livestock farmers worldwide are encouraged to selectively apply treatments against gastrointestinal nematodes (GINs). Targeted selective treatment (TST) of individual animals would be especially useful for smallholder farmers in low-income economies, where cost-effective and sustainable intervention strategies will improve livestock productivity and food security. Supporting research has focused mainly on refining technical indicators for treatment, and much less on factors influencing uptake and effectiveness. We used a mixed method approach, whereby qualitative and quantitative approaches are combined, to develop, implement and validate a TST system for GINs in small ruminants, most commonly goats, among smallholder farmers in the Makgadikgadi Pans region of Botswana, and to seek better understanding of system performance within a cultural context. After the first six months of the study, 42 out of 47 enrolled farmers were followed up; 52% had monitored their animals using the taught inspection criteria and 26% applied TST during this phase. Uptake level showed little correlation with farmer characteristics, such as literacy and size of farm. Herd health significantly improved in those herds where anthelmintic treatment was applied: anaemia, as assessed using the five-point FAMACHA© scale, was 0.44–0.69 points better (95% confidence interval) and body condition score was 0.18–0.36 points better (95% C.I., five-point scale) in treated compared with untreated herds. Only targeting individuals in greatest need led to similar health improvements compared to treating the entire herd, leading to dose savings ranging from 36% to 97%. This study demonstrates that TST against nematodes can be implemented effectively by resource-poor farmers using a community-led approach. The use of mixed methods provides a promising system to integrate technical and social aspects of TST programmes for maximum uptake and effect. PMID:26493540

  19. Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA†

    PubMed Central

    Li, Mei; Duc, Anne-Cécile E.; Klosi, Edvin; Pattabiraman, Srividya; Spaller, Mark R.; Chow, Christine S.

    2009-01-01

    For almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S ribosomal RNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (E. coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (Kds) of the amidated peptide HPVHHYQ-NH2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH2 exhibits moderate affinity for the A-site RNA, with an average Kd value of 16 ?M. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493. PMID:19645415

  20. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques.

    PubMed

    Gowrishankar, Swetha; Yuan, Peng; Wu, Yumei; Schrag, Matthew; Paradise, Summer; Grutzendler, Jaime; De Camilli, Pietro; Ferguson, Shawn M

    2015-07-14

    Through a comprehensive analysis of organellar markers in mouse models of Alzheimer's disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of ?-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer's disease, ?-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular ?-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer's disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology. PMID:26124111

  1. Sulfonoquinovosyl diacylglyceride selectively targets acute lymphoblastic leukemia cells and exerts potent anti-leukemic effects in vivo

    PubMed Central

    Jain, Chetan Kumar; Pradhan, Bhola Shankar; Banerjee, Sukdeb; Mondal, Nirup Bikash; Majumder, Subeer S.; Bhattacharyya, Madhumita; Chakrabarti, Saikat; Roychoudhury, Susanta; Majumder, Hemanta Kumar

    2015-01-01

    DNA topoisomerase II inhibitors e.g. doxorubicin and etoposide are currently used in the chemotherapy for acute lymphoblastic leukemia (ALL). These inhibitors have serious side effects during the chemotherapy e.g. cardiotoxicity and secondary malignancies. In this study we show that sulfonoquinovosyl diacylglyceride (SQDG) isolated from Azadirachta indica exerts potent anti-ALL activity both in vitro and in vivo in nude mice and it synergizes with doxorubicin and etoposide. SQDG selectively targets ALL MOLT-4 cells by inhibiting catalytic activity of topoisomerase I enzyme and inducing p53 dependent apoptotic pathway. SQDG treatment induces recruitment of ATR at chromatin and arrests the cells in S-phase. Down-regulation of topoisomerase I or p53 renders the cells less sensitive for SQDG, while ectopic expression of wild type p53 protein in p53 deficient K562 cells results in chemosensitization of the cells for SQDG. We also show that constant ratio combinations of SQDG and etoposide or SDQG and doxorubicin exert synergistic effects on MOLT-4 cell killing. This study suggests that doses of etoposide/doxorubicin can be substantially reduced by combining SQDG with these agents during ALL chemotherapy and side effects caused can be minimized. Thus dual targeting of topoisomerase I and II enzymes is a promising strategy for improving ALL chemotherapy. PMID:26189912

  2. DC-SIGN-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for HIV disease.

    PubMed

    Gieseler, R K; Marquitan, G; Hahn, M J; Perdon, L A; Driessen, W H P; Sullivan, S M; Scolaro, M J

    2004-05-01

    Myeloid dendritic cells (MyDCs), prime stimulators of antigen-specific immunity, can serve as one of the major reservoirs for human immunodeficiency virus type-1 (HIV-1). Utilizing mature monocyte-derived MyDCs generated with granulocyte/macrophage colony-stimulating factor, interleukin-4, and tumour necrosis factor-alpha as an in vitro model, we here present the first proof of concept for liposomal compound delivery to these cells by specifically addressing CD209, i.e. DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a MyDC-associated C-type lectin implicated in the transmission of HIV-1 to T helper cells. By employing a liposomally entrapped tracer, calcein, we demonstrate by flow cytometry and mathematics a superior targeting efficacy for DC-SIGN, as compared with select other MyDC markers (CD1a, CD4, CD45R0, and CD83). Fluorescence microscopy reveals time-dependent surface binding and intracellular uptake of DC-SIGN-specific liposomes by both immature and mature MyDCs. This pilot study implies that liposomal targeting to CD209 and related C-type lectins may afford therapeutic intracellular drug delivery to MyDCs and other reservoir and nonreservoir cells susceptible to infection with HIV-1. PMID:15140050

  3. Developing a multi-pollutant conceptual framework for the selection and targeting of interventions in water industry catchment management schemes.

    PubMed

    Bloodworth, J W; Holman, I P; Burgess, P J; Gillman, S; Frogbrook, Z; Brown, P

    2015-09-15

    In recent years water companies have started to adopt catchment management to reduce diffuse pollution in drinking water supply areas. The heterogeneity of catchments and the range of pollutants that must be removed to meet the EU Drinking Water Directive (98/83/EC) limits make it difficult to prioritise areas of a catchment for intervention. Thus conceptual frameworks are required that can disaggregate the components of pollutant risk and help water companies make decisions about where to target interventions in their catchments to maximum effect. This paper demonstrates the concept of generalising pollutants in the same framework by reviewing key pollutant processes within a source-mobilisation-delivery context. From this, criteria are developed (with input from water industry professionals involved in catchment management) which highlights the need for a new water industry specific conceptual framework. The new CaRPoW (Catchment Risk to Potable Water) framework uses the Source-Mobilisation-Delivery concept as modular components of risk that work at two scales, source and mobilisation at the field scale and delivery at the catchment scale. Disaggregating pollutant processes permits the main components of risk to be ascertained so that appropriate interventions can be selected. The generic structure also allows for the outputs from different pollutants to be compared so that potential multiple benefits can be identified. CaRPow provides a transferable framework that can be used by water companies to cost-effectively target interventions under current conditions or under scenarios of land use or climate change. PMID:26172105

  4. Diagnosing Lysosomal Storage Disorders: The GM2 Gangliosidoses.

    PubMed

    Hall, Patricia; Minnich, Sara; Teigen, Claire; Raymond, Kimiyo

    2014-01-01

    The GM2 gangliosidoses are a group of autosomal recessive lysosomal storage disorders caused by defective ?-hexosaminidase. There are three clinical conditions in this group: Tay-Sachs disease (TSD), Sandhoff disease (SD), and hexosaminidase activator deficiency. The three conditions are clinically indistinguishable. TSD and SD have been identified with infantile, juvenile, and adult onset forms. The activator deficiency is only known to present with infantile onset. Diagnosis of TSD and SD is based on decreased hexosaminidase activity and a change in the percentage of activity between isoforms. There are no biochemical tests currently available for activator deficiency. This unit provides a detailed procedure for identifying TSD and SD in affected individuals and carriers from leukocyte samples, the most robust sample type available. Curr. Protoc. Hum. Genet. 83:17.16.1-17.16.8. © 2014 by John Wiley & Sons, Inc. PMID:25271840

  5. Newborn screening for lysosomal storage disorders and other neuronopathic conditions.

    PubMed

    Matern, Dietrich; Oglesbee, Devin; Tortorelli, Silvia

    2013-01-01

    Newborn screening (NBS) is a public health program aimed at identifying treatable conditions in presymptomatic newborns to avoid premature mortality, morbidity, and disabilities. Currently, every newborn in the Unites States is screened for at least 29 conditions where evidence suggests that early detection is possible and beneficial. With new or improved treatment options and development of high-throughput screening tests, additional conditions have been proposed for inclusion into NBS programs. Among those are several conditions with a strong neuronopathic component. Some of these conditions have already been added to a few national and international screening programs, whereas others are undergoing pilot studies to determine the test performance metrics. Here, we review the current state of NBS for 13 lysosomal storage disorders, X-adrenoleukodystrophy, Wilson disease, and Friedreich ataxia. PMID:23798012

  6. The heptad repeat region is a major selection target in MERS-CoV and related coronaviruses

    PubMed Central

    Forni, Diego; Filippi, Giulia; Cagliani, Rachele; De Gioia, Luca; Pozzoli, Uberto; Al-Daghri, Nasser; Clerici, Mario; Sironi, Manuela

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) originated in bats and spread to humans via zoonotic transmission from camels. We analyzed the evolution of the spike (S) gene in betacoronaviruses (betaCoVs) isolated from different mammals, in bat coronavirus populations, as well as in MERS-CoV strains from the current outbreak. Results indicated several positively selected sites located in the region comprising the two heptad repeats (HR1 and HR2) and their linker. Two sites (R652 and V1060) were positively selected in the betaCoVs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. During the most recent evolution of MERS-CoV, adaptive mutations in the HR1 (Q/R/H1020) arose in camels or in a previous host and spread to humans. We determined that different residues at position 1020 establish distinct inter- and intra-helical interactions and affect the stability of the six-helix bundle formed by the HRs. A similar effect on stability was observed for a nearby mutation (T1015N) that increases MERS-CoV infection efficiency in vitro. Data herein indicate that the heptad repeat region was a major target of adaptive evolution in MERS-CoV-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function. PMID:26404138

  7. The heptad repeat region is a major selection target in MERS-CoV and related coronaviruses.

    PubMed

    Forni, Diego; Filippi, Giulia; Cagliani, Rachele; De Gioia, Luca; Pozzoli, Uberto; Al-Daghri, Nasser; Clerici, Mario; Sironi, Manuela

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) originated in bats and spread to humans via zoonotic transmission from camels. We analyzed the evolution of the spike (S) gene in betacoronaviruses (betaCoVs) isolated from different mammals, in bat coronavirus populations, as well as in MERS-CoV strains from the current outbreak. Results indicated several positively selected sites located in the region comprising the two heptad repeats (HR1 and HR2) and their linker. Two sites (R652 and V1060) were positively selected in the betaCoVs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. During the most recent evolution of MERS-CoV, adaptive mutations in the HR1 (Q/R/H1020) arose in camels or in a previous host and spread to humans. We determined that different residues at position 1020 establish distinct inter- and intra-helical interactions and affect the stability of the six-helix bundle formed by the HRs. A similar effect on stability was observed for a nearby mutation (T1015N) that increases MERS-CoV infection efficiency in vitro. Data herein indicate that the heptad repeat region was a major target of adaptive evolution in MERS-CoV-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function. PMID:26404138

  8. Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors

    PubMed Central

    Canning, Peter; Tan, Li; Chu, Kiki; Lee, Sam W.; Gray, Nathanael S.; Bullock, Alex N.

    2014-01-01

    The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and ?D helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a ?-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer. PMID:24768818

  9. Selective Photothermolysis to target Sebaceous Glands: Theoretical Estimation of Parameters and Preliminary Results Using a Free Electron Laser

    SciTech Connect

    Fernanda Sakamoto, Apostolos Doukas, William Farinelli, Zeina Tannous, Michelle D. Shinn, Stephen Benson, Gwyn P. Williams, H. Dylla, Richard Anderson

    2011-12-01

    The success of permanent laser hair removal suggests that selective photothermolysis (SP) of sebaceous glands, another part of hair follicles, may also have merit. About 30% of sebum consists of fats with copious CH2 bond content. SP was studied in vitro, using free electron laser (FEL) pulses at an infrared CH2 vibrational absorption wavelength band. Absorption spectra of natural and artificially prepared sebum were measured from 200 nm to 3000 nm, to determine wavelengths potentially able to target sebaceous glands. The Jefferson National Accelerator superconducting FEL was used to measure photothermal excitation of aqueous gels, artificial sebum, pig skin, human scalp and forehead skin (sebaceous sites). In vitro skin samples were exposed to FEL pulses from 1620 to 1720 nm, spot diameter 7-9.5 mm with exposure through a cold 4C sapphire window in contact with the skin. Exposed and control tissue samples were stained using H and E, and nitroblue tetrazolium chloride staining (NBTC) was used to detect thermal denaturation. Natural and artificial sebum both had absorption peaks near 1210, 1728, 1760, 2306 and 2346 nm. Laser-induced heating of artificial sebum was approximately twice that of water at 1710 and 1720 nm, and about 1.5x higher in human sebaceous glands than in water. Thermal camera imaging showed transient focal heating near sebaceous hair follicles. Histologically, skin samples exposed to {approx}1700 nm, {approx}100-125 ms pulses showed evidence of selective thermal damage to sebaceous glands. Sebaceous glands w