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1

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors  

PubMed Central

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

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

2012-01-01

2

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors.  

PubMed

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

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

2012-12-03

3

Selection in favor of lysosomal storage disorders?  

PubMed Central

Four examples of Israeli communities or large families in which high consanguinity is common are presented, with two different lysosomal storage disorders within each community. In each of the four cases the stored substances share common chemical structure, despite the different lysosomal hydrolases involved in each disease. A similar phenomenon is known among the Ashkenazi Jews, in whom four of the most frequent hereditary disorders are lysosomal storage disorders, which are characterized by storage of sphingolipid derivatives. Similar findings are reported in the literature in other communities. We suggest that this phenomenon indicates a selection in favor of lysosomal storage disorders of similar nature in certain populations. The selection forces leading to this phenomenon have not been identified yet, and it has not yet been determined whether these forces are the same in the different communities presented here.

Zlotogora, J; Zeigler, M; Bach, G

1988-01-01

4

Selective release of peptides from lysosomes.  

PubMed

We demonstrate a selective release of peptides by lysosomes in vitro. A lysosomal fraction from human fibroblasts that had previously endocytosed [3H]ribonuclease A was incubated for 2 h, and radioactivity released into the medium and radioactivity retained within lysosomes were analyzed. A variety of radiolabeled molecules including peptides of an appropriate size to serve as antigens for T cell-mediated immunity were released. One small peptide was predominantly released, while others, as well as intact ribonuclease A, were predominantly retained. A 4-5-fold range was also evident in the relative release of three 3H-labeled tripeptide probes of similar charge derived from the sequence of ribonuclease A. This selectivity and the fact that similar peptide degradation fragments were also released and retained by intact cells after endocytosis of [3H]ribonuclease A argues strongly that the release observed in vitro is physiological and not due to damaged lysosomal membranes. PMID:8226924

Isenman, L D; Dice, J F

1993-11-15

5

Lysosomal membrane permeabilization by targeted magnetic nanoparticles in alternating magnetic fields.  

PubMed

Lysosomal death pathways are being explored as alternatives of overcoming cancer tumor resistance to traditional forms of treatment. Nanotechnologies that can selectively target and induce permeabilization of lysosomal compartments in cells could become powerful medical tools. Here we demonstrate that iron oxide magnetic nanoparticles (MNPs) targeted to the epidermal growth factor receptor (EGFR) can selectively induce lysosomal membrane permeabilization (LMP) in cancer cells overexpressing the EGFR under the action of an alternating magnetic field (AMF). LMP was observed to correlate with the production of reactive oxygen species (ROS) and a decrease in tumor cell viability. Confocal microscopy images showed an increase in the cytosolic activity of the lysosomal protease cathepsin B. These observations suggest the possibility of remotely triggering lysosomal death pathways in cancer cells through the administration of MNPs which target lysosomal internalization pathways and the application of AMFs. PMID:23705969

Domenech, Maribella; Marrero-Berrios, Ileana; Torres-Lugo, Madeline; Rinaldi, Carlos

2013-05-24

6

Screening and optimization of ligand conjugates for lysosomal targeting.  

PubMed

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 a good rate of colocalization with 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 the liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared to that with plain liposomes. These results were additionally confirmed by flow cytometry of the intact cells treated with liposomes loaded 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-PEG(2k)-amide and 6-(3-(DSPE-PEG(2k))-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

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

2011-10-06

7

Protein Networks Supporting AP3 Function in Targeting Lysosomal Membrane Proteins  

Microsoft Academic Search

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 triphos- phatase (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

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

2008-01-01

8

Lysosomal Membrane Stabilization and Antiestrogen Action in Specific Hormonal Target Cells (With colour plate I)  

Microsoft Academic Search

Recent advances in these laboratories in analysis of mechanisms of steroid hormone-induced growth have identified lysosomal function in the triggering of the coupled events leading from selective accumulation of the active agent at the cell periphery, to its rapid, cyclic AMP-dependent traversal of the nuclear envelope, and eventual genie activation of the cellular target. Capture of trophic steroid hormone leads

Clara M. Szego

1972-01-01

9

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.  

PubMed Central

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes. Images Fig.1 Fig.3 Fig.4 Fig.5 Fig.6 Fig.8 Fig.9 Fig.10 Fig.11

Peters, C; Braun, M; Weber, B; Wendland, M; Schmidt, B; Pohlmann, R; Waheed, A; von Figura, K

1990-01-01

10

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

Microsoft Academic Search

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

Ana Maria Cuervo

2011-01-01

11

Targeting of lysosomes by liposomes modified with octadecyl-rhodamine B.  

PubMed

The use of lysosome-targeted liposomes may significantly improve a delivery of therapeutic enzymes into lysosomes for the treatment of lysosome-associated diseases. The aim of this research was to achieve a specific intracellular targeting of lysosomes, by using liposomes modified with the lysosomotropic octadecyl-rhodamine B (RhB) and loaded with a model compound, fluorescein isothiocyanate (FITC)-dextran (FD). Plain and RhB-modified liposomes were prepared by hydration of lipid films and loaded with FD or with 5-dodecanoylaminofluorescein di-?-d-galactopyranoside (C(12)FDG), a specific substrate for the intralysosomal ?-galactosidase. The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal microscopy, flow cytometry, and subcellular fractionation. Confocal microscopy demonstrated that RhB-liposomes co-localize well with the specific lysosomal markers, unlike plain liposomes. The comparison of the FITC fluorescence of the lysosomes isolated by subcellular fractionation also showed that the efficiency of FD delivery into lysosomes by RhB-modified liposomes was significantly higher compared with plain liposomes. These results were additionally confirmed by the flow cytometry of the intact cells treated with C(12)FDG-loaded liposomes that also demonstrated increased lysosomal targeting by RhB-modified liposomes. The modification of the liposomal surface with a lysosomotropic ligand, such as octadecyl-RhB, can significantly increase the delivery of liposomal loads to lysosomes. PMID:21275828

Koshkaryev, Alexander; Thekkedath, Ritesh; Pagano, Cinzia; Meerovich, Igor; Torchilin, Vladimir P

2011-01-29

12

A Receptor for the Selective Uptake and Degradation of Proteins by Lysosomes  

NASA Astrophysics Data System (ADS)

Multiple pathways of protein degradation operate within cells. A selective protein import pathway exists for the uptake and degradation of particular cytosolic proteins by lysosomes. Here, the lysosomal membrane glycoprotein LGP96 was identified as a receptor for the selective import and degradation of proteins within lysosomes. Specific substrates of this proteolytic pathway bound to the cytosolic tail of a 96-kilodalton lysosomal membrane protein in two different binding assays. Overexpression of human LGP96 in Chinese hamster ovary cells increased the activity of the selective lysosomal proteolytic pathway in vivo and in vitro.

Cuervo, Ana Maria; Dice, J. Fred

1996-07-01

13

Proteasome Inhibitors Block a Late Step in Lysosomal Transport of Selected Membrane but not Soluble Proteins  

Microsoft Academic Search

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin- proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands,

Peter van Kerkhof; Cristina M. Alves dos Santos; Martin Sachse; Judith Klumperman; Guojun Bu; Ger J. Strous

2001-01-01

14

Intracellular target for alpha-terthienyl photosensitization: involvement of lysosomal membrane damage.  

PubMed

Intracellular targets for the photosensitizer alpha-terthienyl (alpha T) were examined by fluorescence microscopy and microfluorospectrometry using human nonkeratinized buccal cells. Intracellular distribution of alpha T was observed as fluorescent patches widely dispersed in the cytoplasm. The distribution of the fluorescent patches was compared with that of acid phosphatase activity visualized as an azo dye produced by the fast garnet 2-methyl-4-[(2-methyl-phenyl)azo]benzenediasonium sulfate reaction. Because both the distribution sites coincided, lysosomes were the likely sites of intracellular affinity of alpha T. However, because acid phosphatase is not a specific lysosomal marker, we tried to detect another lysosomal enzyme, beta-galactosidase, to confirm if the fluorescent patches were lysosomes, using fluorescein-di-(beta-D-galactopyranoside) (FDG) as a fluorogenic substrate. Without UV-A (320-400 nm) irradiation of the cells after uptake of alpha T and FDG, no significant fluorescence was observed. In contrast, with prior UV-A irradiation in the presence of alpha T and FDG, the bright yellow fluorescence of fluorescein, which is the digested product of FDG, was clearly detected in the cells by fluorescence microscopy. This observation implied that inflow of external FDG into the lysosomes is caused by lysosomal membrane damage on alpha T photosensitization. The present results indicated that lysosomes are the primary photosensitization site of alpha T. PMID:8337250

Sasaki, M; Koyama, S; Tokiwa, K; Fujita, H

1993-05-01

15

Generation and characterization of a lysosomally targeted, genetically encoded Ca2+-sensor  

PubMed Central

Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide ‘trigger’ release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

McCue, Hannah V.; Wardyn, Joanna D.; Burgoyne, Robert D.; Haynes, Lee P.

2012-01-01

16

Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice  

Microsoft Academic Search

Enzyme-replacement therapy is an established means of treating lysosomal storage diseases. Infused therapeutic enzymes are targeted to lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate, on the enzymes. We have tested an alternative, peptide-based targeting system for delivery of enzymes to lysosomes in a murine mucopolysaccharidosis type VII (MPS

Jonathan H. Lebowitz; Jeffrey H. Grubb; John A. Maga; Deborah H. Schmiel; Carole Vogler; William S. Sly

2004-01-01

17

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

SciTech Connect

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.

Haylett, T.; Thilo, L.

1986-10-01

18

Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion  

PubMed Central

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity- purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

1992-01-01

19

SETI target selection  

NASA Astrophysics Data System (ADS)

The NASA high resolution microwave survey (HRMS) consists of two elements, a survey of the entire sky and a targeted search of nearby stars which is much more sensitive. Strategies are proposed for target selection for the targeted search with goals of improving the chances of successful detection of signals from technological civilizations that inhabit planets around solar-type stars, and to minimize the chances of missing signals from unexpected sites.

Latham, David W.; Soderblom, David R.

1993-10-01

20

Lysosomes as a possible target of enniatin B-induced toxicity in Caco-2 cells.  

PubMed

Enniatins are cyclic hexadepsipeptidic mycotoxins with ionophoric, antibiotic, and insecticidal activity. Enniatin B (EnnB), the most important analogue, is produced by many Fusarium species and is a common contaminant in grain-based foods. The compound's cytotoxic potential has been shown in different experiments; however, the mode of action has not been detailed so far. In the present study, several mutually confirmative experiments have been performed indicating that EnnB-initiated cytotoxicity could be connected with lysosomal membrane permeabilization (LMP). Lysosomal functionality, as assessed by the Neutral Red assay, was already affected after 3 h of toxin exposure. After 24 h, cell proliferation was decreased, and there was indication for a cell cycle arrest in the G(2)/M phase leading to the initiation of apoptosis or necrosis. Intracellular ROS-production was observed. However, antioxidants did not alter the observed EnnB-induced loss of lysosomal functionality leading to the conclusion that ROS was not an initial factor but one produced later in the event cascade. The collected data suggested that lysosomal destabilization is an upstream event in EnnB-initiated cytotoxicity followed by a certain extent of translocation of cathepsins into the cytosol, which was observed using immunological and proteomic methods. It appeared that cell death induced by EnnB was delayed and occurred not as a massive lysosomal breakdown but was probably progressing and leading to partial and selective LMP, starting a nonapoptotic cell death pathway with morphological features that had been previously considered as necrotic. The molecular mechanism of EnnB-triggered lysosomal destabilization, and the cellular processes leading to mitochondrial permeabilization and cell death are still unknown. They may, however, be connected to the compound's ionophoric properties. PMID:22731695

Ivanova, L; Egge-Jacobsen, W M; Solhaug, A; Thoen, E; Fæste, C K

2012-07-19

21

SETI target selection.  

NASA Astrophysics Data System (ADS)

The NASA High Resolution Microwave Survey consists of two complementary elements: a Sky Survey of the entire sky to a moderate level of sensitivity; and a Targeted Search of nearby stars, one at a time, to a much deeper level of sensitivity. The authors propose strategies for target selection with two goals: to improve the chances of successful detection of signals from technical civilizations that inhabit planets around solar-type stars, and to minimize the chances of missing signals from unexpected sites.

Latham, D. W.; Soderblom, D. R.

1995-06-01

22

An Intralysosomal hsp70 Is Required for a Selective Pathway of Lysosomal Protein Degradation  

Microsoft Academic Search

Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimu- lating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect im- munofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of

Fernando A. Agarraberes; Stanley R. Terlecky; J. Fred Dice

1997-01-01

23

Peptide sequences that target proteins to lysosomes for enhanced degradation during serum withdrawal  

SciTech Connect

Ribonuclease A (RNase A) microinjected into human diploid fibroblasts is degraded with a half-life of 80-100 h in the presence of serum, but its half-life declines to 40 h when cells are deprived of serum. This increased degradation in response to serum withdrawal results from an increased rate of uptake of microinjected RNase A from the cytosol into lysosomes. The cells' ability to recognize RNase A for this enhanced degradation is based on some feature of amino acids 1-20. Covalent linkage of S-peptide to other proteins results in enhanced lysosomal degradation of the conjugate in response to serum withdrawal. The authors have further defined the essential region of RNase S-peptide to be within residues 7-11, KFERQ. Coinjection of radiolabeled RNase A and excess unlabeled pentapeptide specifically blocks the enhanced degradation of RNase A. Affinity-purified polyclonal IgGs raised against KFERQ immunoprecipitate 25-30% of radiolabeled cytosolic proteins from human fibroblasts. Pulse-chase experiments indicate that these proteins are preferentially degraded upon serum withdrawal while degradation of nonimmuno-precipitated proteins is unaffected. These results suggest that peptide sequences similar to KFERQ are contained within cellular proteins and that such sequences target proteins to lysosomes for enhanced degradation during serum withdrawal.

Dice, J.F.; Backer, J.M.; Chiang, H.L.

1987-05-01

24

SETI target selection.  

PubMed

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

Latham, D W; Soderblom, D R

25

Role of Adaptor Complex AP3 in Targeting Wild-Type and Mutated CD63 to Lysosomes  

Microsoft Academic Search

CD63 is a lysosomal membrane protein that belongs to the tetraspanin family. Its carboxyterminal cytoplasmic tail sequence contains the lysosomal targeting motif GYEVM. Strong, tyrosine- dependent interaction of the wild-type carboxyterminal tail of CD63 with the AP-3 adaptor subunit 3 was observed using a yeast two-hybrid system. The strength of interaction of mutated tail sequences with 3 correlated with the

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

2002-01-01

26

Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes  

PubMed Central

Mouse embryonic fibroblasts that are deficient in the two mannose 6- phosphate receptors (MPRs) MPR 46 and MPR 300 missort the majority (> or = 85%) of soluble lysosomal proteins into the medium. Human MPR 46 and MPR 300 were expressed in these cells to test whether overexpression of a single type of MPR can restore transport of lysosomal proteins to lysosomes. Only a partial correction of the missorting was observed after overexpression of MPR 46. Even at MPR 46 levels that are five times higher than the wild-type level, more than one third of the newly synthesized lysosomal proteins accumulates in the secretions. Two-fold overexpression of MPR 300 completely corrects the missorting of lysosomal enzymes. However, at least one fourth of the lysosomal enzymes are transported along a secretion-recapture pathway that is sensitive to mannose 6-phosphate in medium. In control fibroblasts that express both types of MPR, the secretion-recapture pathway is of minor importance. These results imply that neither overexpression of MPR 46 nor MPR 300 is sufficient for targeting of lysosomal proteins along intracellular routes.

1996-01-01

27

Lysosomes and lysosomal cathepsins in cell death.  

PubMed

Lysosomes are the key degradative compartments of the cell. Lysosomal cathepsins, which are enclosed in the lysosomes, help to maintain the homeostasis of the cell's metabolism by participating in the degradation of heterophagic and autophagic material. Following the targeted lysosomal membrane's destabilization, the cathepsins can be released into the cytosol and initiate the lysosomal pathway of apoptosis through the cleavage of Bid and the degradation of the anti-apoptotic Bcl-2 homologues. Cathepsins can also amplify the apoptotic signaling, when the lysosomal membranes are destabilized at a later stage of apoptosis, initiated by other stimuli. However, the functional integrity of the lysosomal compartment during apoptosis enables efficient autophagy, which can counteract apoptosis by providing the energy source and by disposing the damaged mitochondria, which generate the ROS. Impairing autophagy by disabling the lysosome function is being investigated as an adjuvant therapeutic approach to sensitize cells to apoptosis-inducing agents. Destabilization of the lysosomal membranes by the lysosomotropic detergents seems to be a promising strategy in this context as it would not only disable autophagy, but also promote apoptosis through the initiation of the lysosomal pathway. In contrast, the impaired autophagy and lysosomal degradation linked with the increased oxidative stress underlie degenerative changes in the aging neurons. This further suggests that lysosomes and lysosomal cathepsins have a dual role in cell death. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. PMID:21914490

Repnik, Urška; Stoka, Veronika; Turk, Vito; Turk, Boris

2011-09-03

28

Targeting HER2+ breast cancer cells: Lysosomal accumulation of anti-HER2 antibodies is influenced by antibody binding site and conjugation to polymeric nanoparticles.  

PubMed

Humanized monoclonal antibodies (mAb) against HER2 are being engineered to treat cancer. We utilized phage-display technology to generate a novel anti-HER2 mAb (named 73JIgG) that binds an epitope of HER2 distinct from that of trastuzumab. Although these mAbs bind to the same cell surface receptor, they have different cell distribution profiles. After 3h of incubation, almost 10% of the total 73JIgG reaches the lysosome compared to less than 3% of trastuzumab. Interestingly, 73JIgG disassociates from HER2 whereas trastuzumab remains bound to the receptor. Importantly, HER2 distribution is not affected by the antibody binding epitope, thus negating this mechanism as the reason for the difference in intracellular trafficking of 73JIgG versus trastuzumab. Each of trastuzumab and 73JIgG was chemically-modified with either a small molecule or polymeric nanoparticle to better understand the influence of conjugation on cellular localization. Relative to antibody alone, antibody-nanoparticle conjugates resulted in a higher concentration of antibodies in the lysosome whereas antibody-small molecule conjugates did not affect cell trafficking to the lysosome. Given the importance of lysosomal targeting, these results demonstrate the importance of understanding the influence of the antibody-conjugate on cell trafficking for ultimate optimization of treatment selection. PMID:23880472

Owen, Shawn C; Patel, Nish; Logie, Jennifer; Pan, Guohua; Persson, Helena; Moffat, Jason; Sidhu, Sachdev S; Shoichet, Molly S

2013-07-21

29

A Requirement for Bid for Induction of Apoptosis by Photodynamic Therapy with a Lysosome- but not a Mitochondrion-Targeted Photosensitizer  

PubMed Central

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photo-disrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid?/? cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.

Chiu, Song-mao; Xue, Liang-yan; Lam, Minh; Rodriguez, Myriam E.; Zhang, Ping; Kenney, Malcolm E.; Nieminen, Anna-Liisa; Oleinick, Nancy L.

2010-01-01

30

Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy  

PubMed Central

In the hallmark neuritic dystrophy of Alzheimer’s disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers, Rab7 and LAMP1, and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated upon fusion with bi-directionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, APP, and neurofilament protein hyperphosphorylation. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn, largely cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD.

Lee, Sooyeon; Sato, Yutaka; Nixon, Ralph A.

2012-01-01

31

Targeting macrophages with baculovirus-produced lysosomal enzymes: implications for enzyme replacement therapy of the glycoprotein storage disorder galactosialidosis  

Microsoft Academic Search

Lysosomal storage diseases (LSDs) are monogenic disorders of metabolism caused by deficiency of hydrolytic enzymes. In several LSDs, cells of the reticuloendothelial (RE) system are the primary targets of the disease. Exogenous administration of recombinant enzymes overproduced in mammalian cells has proved effective for treating the systemic phenotype in nonneuropathic patients with LSDs. However, for the treatment of diseases with

Erik J. Bonten; Dongning Wang; James N. Toy; Linda Mann; Aurélie Mignardot; Gouri Yogalingam; Alessandra d'Azzo

2004-01-01

32

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

PubMed

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

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

2012-12-10

33

Differential Requirements for Actin Polymerization, Calmodulin, and Ca2+ Define Distinct Stages of Lysosome/Phagosome Targeting  

PubMed Central

Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies, and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process, we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. Incubation of tritium-labeled lysosomes with phagosomes containing scintillant latex beads led to light emission in a reaction requiring cytosol, ATP, and low Ca2+ concentrations. The nascent complex was sensitive to disruption by alkaline carbonate, indicating that the organelles had “docked” but not fused. Through inhibitor studies and fluorescence microscopy we show that docking is preceded by a tethering step that requires actin polymerization and calmodulin. In the docked state ongoing actin polymerization and calmodulin are no longer necessary. The tethering/docking activity was purified to near homogeneity from rat liver cytosol. Major proteins in the active fractions included actin, calmodulin and IQGAP2. IQGAPs are known to bind calmodulin and cross-link F-actin, suggesting a key coordinating role during lysosome/phagosome attachment. The current results support the conclusion that lysosome/phagosome interactions proceed through distinct stages and provide a useful new approach for further experimental dissection.

Stockinger, Walter; Zhang, Shao C.; Trivedi, Vishal; Jarzylo, Larissa A.; Shieh, Eugenie C.; Lane, William S.; Castoreno, Adam B.; Nohturfft, Axel

2006-01-01

34

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

PubMed Central

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

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

2011-01-01

35

TPC Proteins Are Phosphoinositide-activated Sodium-selective Ion Channels in Endosomes and Lysosomes  

PubMed Central

Summary Mammalian Two-Pore Channels (TPC1, 2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P2, and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na+, not K+, as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes, and may explain the specificity of PI(3,5)P2 in regulating the fusogenic potential of intracellular organelles.

Wang, Xiang; Zhang, Xiaoli; Dong, Xian-ping; Samie, Mohammad; Li, Xinran; Cheng, Xiping; Goschka, Andrew; Shen, Dongbiao; Zhou, Yandong; Harlow, Janice; Zhu, Michael X.; Clapham, David E.; Ren, Dejian; Xu, Haoxing

2012-01-01

36

IkB Is a Substrate for a Selective Pathway of Lysosomal Proteolysis  

Microsoft Academic Search

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor k B( IkB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IkB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IkB is directly transported into isolated

Ana Maria Cuervo; Wei Hu; Bing Lim; J. Fred Dice

2010-01-01

37

Inactivation of the {beta}-subunit of lysosomal {beta}-hexosaminidase by targeted disruption of the HEXB gene in mice  

SciTech Connect

{beta}-Hexosaminidase is a lysosomal enzyme that catalyzes the hydrolysis of ganglioside G{sub M2} and other substances. Two major catalytically active isozymes are expressed in human tissues: Hex A (a{beta}) and Hex B ({beta}{beta}). Sandhoff disease is a rare autosomal recessive disorder caused by mutations in the HEXB gene, inactivating both isoenzymes. We have characterized the structure of the mouse HEXB gene from the strain 129/Sv. It contains 14 exons in a span of 22 kb; a 1200 by 5{prime}-sequence showed promoter activity when expressed in monkey COS cells. A survey of mRNA levels in mouse tissues showed the highest levels in kidney and epididymis, with brain intermediate, and lung, testis and liver with the lowest levels. We used homologous recombination of a distrupted HEXB gene to inactivate the endogenous gene in pluripotent embryonic stem (ES) cells. The sequence replacement vector contained {approximately}11 kb of the mouse HEXB gene (exons 1-5) and was interrupted in the second exon with the neomycin-resistance (neo{sup r}) gene. The targeting vector was introduced into male CGR8 ES cells by electroporation followed by selection for G418-resistant colonies. Analysis of the genomic DNA of 100 independent colonies by Southern blot allowed us to identify six cell lines with the expected band pattern generated by specific recombination at the targeted site. Cells of three of the cell lines were microinjected into C57BL/6J blastocysts in order to generate chimeric mice. Three chimeric males were obtained with ES cell contributions ranging from 80 to 95% as judged by the proportion of agouti coat color. These chimeras are currently being bred with C57BL/6J mice for the generation of HEXB heterozygous and homozygous mice.

Phaneuf, D.; Trasler, J.; Gravel, R.A. [McGill Univ., Quebec (Canada)] [and others

1994-09-01

38

Chaperone-mediated autophagy targets hypoxia-inducible factor-1? (HIF-1?) for lysosomal degradation.  

PubMed

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that mediates adaptive responses to hypoxia. We demonstrate that lysosomal degradation of the HIF-1? subunit by chaperone-mediated autophagy (CMA) is a major regulator of HIF-1 activity. Pharmacological inhibitors of lysosomal degradation, such as bafilomycin and chloroquine, increased HIF-1? levels and HIF-1 activity, whereas activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased HIF-1? levels and HIF-1 activity. In contrast, macroautophagy inhibitors did not increase HIF-1 activity. Transcription factor EB, a master regulator of lysosomal biogenesis, also negatively regulated HIF-1 activity. HIF-1? interacts with HSC70 and LAMP2A, which are core components of the CMA machinery. Overexpression of HSC70 or LAMP2A decreased HIF-1? protein levels, whereas knockdown had the opposite effect. Finally, hypoxia increased the transcription of genes involved in CMA and lysosomal biogenesis in cancer cells. Thus, pharmacological and genetic approaches identify CMA as a major regulator of HIF-1 activity and identify interplay between autophagy and the response to hypoxia. PMID:23457305

Hubbi, Maimon E; Hu, Hongxia; Kshitiz; Ahmed, Ishrat; Levchenko, Andre; Semenza, Gregg L

2013-03-01

39

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

PubMed Central

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

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

2007-01-01

40

Poly(L-aspartic acid) nanogels for lysosome-selective antitumor drug delivery.  

PubMed

Advanced materials that have controllable pH-responsive properties when submerged in the lysosome have a great potential in intracellular drug delivery. We developed novel poly(L-amino acid) nanogels that were prepared by a facile cross-linking of poly[L-aspartic acid-g-(3-diethylaminopropyl)]-b-poly(ethylene glycol)-maleimide [poly(L-Asp-g-DEAP)-b-PEG-Mal] and poly(L-aspartic acid-g-ethyl thiol)-b-PEG [poly(L-Asp-SH)-b-PEG] in an oil/water emulsion condition. Interestingly, these nanogels (~125 nm in diameter) modulated volume expansion (~375 nm in diameter) in a lysosomal pH (~pH 5.0) due to an extensive proton absorption of DEAP at a low pH, which mediated lysosome swelling and the subsequent lysosome destabilization. In the in vitro tumor cell cytotoxicity test, they encouraged tumor cell death, probably owing to the leakage of lysosomal enzymes. Furthermore, encapsulating antitumor drug (e.g., doxorubicin, DOX) into these nanogels enhanced tumor cell cytotoxicity. We conclude that this nanogel system will have great potential for tumor therapy. PMID:23010033

Oh, Nam Muk; Oh, Kyung Taek; Youn, Yu Seok; Lee, Deok-Keun; Cha, Kyung-Hoi; Lee, Don Haeng; Lee, Eun Seong

2012-07-20

41

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum.  

PubMed

Most intracellular Ca(2+) signals result from opening of Ca(2+) channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca(2+) into mitochondria. Ca(2+) channels in lysosomes contribute to endo-lysosomal trafficking and Ca(2+) signalling, but the role of lysosomal Ca(2+) uptake in Ca(2+) signalling is unexplored. Inhibition of lysosomal Ca(2+) uptake by dissipating the H(+) gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca(2+) signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation. Bafilomycin A1 amplified the Ca(2+) signals evoked by photolysis of caged Ins(1,4,5)P(3) or by inhibition of ER Ca(2+) pumps, and it slowed recovery from them. Ca(2+) signals evoked by store-operated Ca(2+) entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca(2+) released by Ins(1,4,5)P(3) receptors. PMID:23097044

López-Sanjurjo, Cristina I; Tovey, Stephen C; Prole, David L; Taylor, Colin W

2012-10-24

42

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum  

PubMed Central

Summary Most intracellular Ca2+ signals result from opening of Ca2+ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca2+ into mitochondria. Ca2+ channels in lysosomes contribute to endo-lysosomal trafficking and Ca2+ signalling, but the role of lysosomal Ca2+ uptake in Ca2+ signalling is unexplored. Inhibition of lysosomal Ca2+ uptake by dissipating the H+ gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca2+ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation. Bafilomycin A1 amplified the Ca2+ signals evoked by photolysis of caged Ins(1,4,5)P3 or by inhibition of ER Ca2+ pumps, and it slowed recovery from them. Ca2+ signals evoked by store-operated Ca2+ entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca2+ released by Ins(1,4,5)P3 receptors.

Lopez-Sanjurjo, Cristina I.; Tovey, Stephen C.; Prole, David L.; Taylor, Colin W.

2013-01-01

43

Ethical Implications of Target Market Selection  

Microsoft Academic Search

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

Terri L. Rittenburg; Madhavan Parthasarathy

1997-01-01

44

Chemical principles for the design of a novel fluorescent probe with high cancer-targeting selectivity and sensitivity.  

PubMed

Understanding of principles governing selective and sensitive cancer targeting is critical for development of chemicals for cancer diagnostics and treatment. We determined the underlying mechanisms of how a novel fluorescent small organic molecule, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), selectively labels cancer cells but not normal cells. We show that BMVC is retained in the lysosomes of normal cells. In cancer cells, BMVC escapes lysosomal retention and localizes to the mitochondria or to the nucleus, where DNA-binding dramatically increases BMVC fluorescence intensity, allowing it to light up only cancer cells. Structure-function analyses of BMVC derivatives show that hydrogen-bonding capacity is a key determinant of lysosomal retention in normal cells, whereas lipophilicity directs these derivatives to the mitochondria or the nucleus in cancer cells. In addition, drug-resistant cancer cells preferentially retain BMVC in their lysosomes compared to drug-sensitive cancer cells, and BMVC can be released from drug-resistant lysosomes using lysosomotropic agents. Our results further our understanding of how properties of cellular organelles differ between normal and cancer cells, which can be exploited for diagnostic and/or therapeutic use. We also provide physiochemical design principles for selective targeting of small molecules to different organelles. Moreover, our results suggest that agents which can increase lysosomal membrane permeability may re-sensitize drug-resistant cancer cells to chemotherapeutic agents. PMID:23970166

Kang, Chi-Chih; Huang, Wei-Chun; Kouh, Chiung-Wen; Wang, Zi-Fu; Cho, Chih-Chien; Chang, Cheng-Chung; Wang, Chiung-Lin; Chang, Ta-Chau; Seemann, Joachim; Huang, Lily Jun-Shen

2013-09-23

45

Overexpression of Human a-Galactosidase A Results in Its Intracellular Aggregation, Crystallization in Lysosomes, and Selective Secretion  

Microsoft Academic Search

Human lysosomal oe-galactosidase A (o~-Gal A) was stably overexpressed in CHO cells and its bio- synthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme over- expressor, produced intracellular ot-Gal A levels of 20,900 U\\/mg ('~100 gg of enzyme\\/107 cells) and secreted ~13,000 U (or 75\\/~g\\/107 cells) per day. U1- trastructural examination of these ceils revealed numerous 0.25-1.5

Yiarmis A. Ioannou; David E Bishop; Robert J. Desnick

1992-01-01

46

Selectively targeting estrogen receptors for cancer treatment  

PubMed Central

Estrogens regulate growth and development through the action of two distinct estrogen receptors (ERs), ER? and ER?, which mediate proliferation and differentiation of cells. For decades, ER? mediated estrogen signaling has been therapeutically targeted to treat breast cancer, most notably with the selective estrogen receptor modulator (SERM) tamoxifen. Selectively targeting ERs occurs at two levels: tissue selectivity and receptor subtype selectivity. SERMs have been developed with emphasis on tissue selectivity to target ER signaling for breast cancer treatment. Additionally, new approaches to selectively target the action of ER? going beyond ligand-dependent activity are under current investigation. As evidence of the anti-proliferative role of ER? accumulates, selectively targeting ER? is an attractive approach for designing new cancer therapies with the emphasis shifted to designing ligands with subtype selectivity. This review will present the mechanistic and structural features of ERs that determine tissue and subtype selectivity with an emphasis on current approaches to selectively target ER? and ER? for cancer treatment.

Shanle, Erin K.; Xu, Wei

2010-01-01

47

Quasar target selection fiber efficiency  

SciTech Connect

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

Newberg, H.; Yanny, B.

1996-05-01

48

BODIPY-labeled DC-SIGN-targeting glycodendrons efficiently internalize and route to lysosomes in human dendritic cells.  

PubMed

Glycodendrons bearing nine copies of mannoses or fucoses have been prepared by an efficient convergent strategy based on Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC). These glycodendrons present a well-defined structure and have an adequate size and shape to interact efficiently with the C-type lectin DC-SIGN. We have selected a BODIPY derivative to label these glycodendrons due to its interesting physical and chemical properties as chromophore. These BODIPY-labeled glycodendrons were internalized into dendritic cells by mean of DC-SIGN. The internalized mannosylated and fucosylated dendrons are colocalized with LAMP1, which suggests routing to lysosomes. The interaction of these glycodendrons with DC-SIGN at the surface of dendritic cells did not induce maturation of the cells. Signaling analysis by checking different cytokines indicated also the lack of induction the expression of inflammatory and noninflammatory cytokines by these second generation glycodendrons. PMID:22920925

Ribeiro-Viana, Renato; García-Vallejo, Juan J; Collado, Daniel; Pérez-Inestrosa, Ezequiel; Bloem, Karien; van Kooyk, Yvette; Rojo, Javier

2012-09-07

49

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

Microsoft Academic Search

Enzyme replacement therapies for lysosomal storage disorders are often hindered by suboptimal biodistribution of recombinant enzymes after systemic injection. This is the case for Pompe disease caused by acid ?-glucosidase (GAA) deficiency, leading to excess glycogen storage throughout the body, mainly the liver and striated muscle. Targeting intercellular adhesion molecule-1 (ICAM-1), a protein involved in inflammation and overexpressed on most

Janet Hsu; Laura Northrup; Tridib Bhowmick; Silvia Muro

50

Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function  

Microsoft Academic Search

Lysosomes are the primary catabolic compartments of eukaryotic cells. They degrade extracellular material that has been internalized by endocytosis and intracellular components that have been sequestered by autophagy. In addition, specialized cells contain lysosome-related organelles that store and secrete proteins for cell-type-specific functions. The functioning of a healthy cell is dependent on the proper targeting of newly synthesized lysosomal proteins.

Paul Saftig; Judith Klumperman

2009-01-01

51

Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes.  

PubMed

Cationic amphiphilic drugs (CADs) cause massive intracellular accumulation of phospholipids, thereby resulting in phospholipidosis (PLD); however, the molecular mechanism underlying CAD-induced PLD remains to be resolved. Here, we found that treatment of normal rat kidney cells with CADs known to induce PLD caused redistribution of a mannose 6-phosphate/IGF-II receptor (MPR300) from the TGN to endosomes and concomitantly increased the secretion of lysosomal enzymes, resulting in a decline of intracellular lysosomal enzyme levels. These results enable the interpretation of why CADs cause excessive accumulation of undegraded substrates, including phospholipids in lysosomes, and led to the conclusion that the impaired MPR300-mediated sorting system of lysosomal enzymes reflects the general mechanism of CAD-induced PLD. In addition, our findings suggest that the measurement of lysosomal enzyme activity secreted into culture medium is useful as a rapid and convenient in vitro early screening system to predict drugs that can induce PLD. PMID:18840403

Ikeda, Kazuhiko; Hirayama, Masahiro; Hirota, Yuko; Asa, Erika; Seki, Jiro; Tanaka, Yoshitaka

2008-10-07

52

Comparative binding, endocytosis, and biodistribution of antibodies and antibody-coated carriers for targeted delivery of lysosomal enzymes to ICAM-1 versus transferrin receptor.  

PubMed

Targeting lysosomal enzymes to receptors involved in transport into and across cells holds promise to enhance peripheral and brain delivery of enzyme replacement therapies (ERTs) for lysosomal storage disorders. Receptors being explored include those associated with clathrin-mediated pathways, yet other pathways seem also viable. Well characterized examples are that of transferrin receptor (TfR) and intercellular adhesion molecule 1 (ICAM-1), involved in iron transport and leukocyte extravasation, respectively. TfR and ICAM-1 support ERT delivery via clathrin- vs. cell adhesion molecule-mediated mechanisms, displaying different valency and size restrictions. To comparatively assess this, we used antibodies vs. larger multivalent antibody-coated carriers and evaluated TfR vs. ICAM-1 binding and endocytosis in endothelial cells, as well as in vivo biodistribution and delivery of a model lysosomal enzyme required in peripheral organs and brain: acid sphingomyelinase (ASM), deficient in types A-B Niemann Pick disease. We found similar binding of antibodies to both receptors under control conditions, with enhanced binding to activated endothelium for ICAM-1, yet only anti-TfR induced endocytosis efficiently. Contrarily, antibody-coated carriers showed enhanced binding, engulfment, and endocytosis for ICAM-1. In mice, anti-TfR enhanced brain targeting over anti-ICAM, with an opposite outcome in the lungs, while carriers enhanced ICAM-1 targeting over TfR in both organs. Both targeted carriers enhanced ASM delivery to the brain and lungs vs. free ASM, with greater enhancement for anti-ICAM carriers. Therefore, targeting TfR or ICAM-1 improves lysosomal enzyme delivery. Yet, TfR targeting may be more efficient for smaller conjugates or fusion proteins, while ICAM-1 targeting seems superior for multivalent carrier formulations. PMID:22968581

Papademetriou, Jason; Garnacho, Carmen; Serrano, Daniel; Bhowmick, Tridib; Schuchman, Edward H; Muro, Silvia

2012-09-12

53

Secretory lysosomes  

Microsoft Academic Search

Regulated secretion of stored secretory products is important in many cell types. In contrast to professional secretory cells, which store their secretory products in specialized secretory granules, some secretory cells store their secretory proteins in a dual-function organelle, called a secretory lysosome. Functionally, secretory lysosomes are unusual in that they serve both as a degradative and as a secretory compartment.

Emma J. Blott; Gillian M. Griffiths

2002-01-01

54

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

PubMed Central

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

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

2013-01-01

55

Characterization of proprotein convertase subtilisin/kexin type 9 (PCSK9) trafficking reveals a novel lysosomal targeting mechanism via amyloid precursor-like protein 2 (APLP2).  

PubMed

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

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

2013-02-19

56

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

PubMed

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

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

2013-09-01

57

Absence of ?-Interferon-inducible Lysosomal Thiol Reductase in Melanomas Disrupts T Cell Recognition of Select Immunodominant Epitopes  

PubMed Central

Long-lasting tumor immunity requires functional mobilization of CD8+ and CD4+ T lymphocytes. CD4+ T cell activation is enhanced by presentation of shed tumor antigens by professional antigen-presenting cells (APCs), coupled with display of similar antigenic epitopes by major histocompatibility complex class II on malignant cells. APCs readily processed and presented several self-antigens, yet T cell responses to these proteins were absent or reduced in the context of class II+ melanomas. T cell recognition of select exogenous and endogenous epitopes was dependent on tumor cell expression of ?-interferon–inducible lysosomal thiol reductase (GILT). The absence of GILT in melanomas altered antigen processing and the hierarchy of immunodominant epitope presentation. Mass spectral analysis also revealed GILT's ability to reduce cysteinylated epitopes. Such disparities in the profile of antigenic epitopes displayed by tumors and bystander APCs may contribute to tumor cell survival in the face of immunological defenses.

Haque, M. Azizul; Li, Ping; Jackson, Sheila K.; Zarour, Hassane M.; Hawes, John W.; Phan, Uyen T.; Maric, Maja; Cresswell, Peter; Blum, Janice S.

2002-01-01

58

Absence of gamma-interferon-inducible lysosomal thiol reductase in melanomas disrupts T cell recognition of select immunodominant epitopes.  

PubMed

Long-lasting tumor immunity requires functional mobilization of CD8+ and CD4+ T lymphocytes. CD4+ T cell activation is enhanced by presentation of shed tumor antigens by professional antigen-presenting cells (APCs), coupled with display of similar antigenic epitopes by major histocompatibility complex class II on malignant cells. APCs readily processed and presented several self-antigens, yet T cell responses to these proteins were absent or reduced in the context of class II+ melanomas. T cell recognition of select exogenous and endogenous epitopes was dependent on tumor cell expression of gamma-interferon-inducible lysosomal thiol reductase (GILT). The absence of GILT in melanomas altered antigen processing and the hierarchy of immunodominant epitope presentation. Mass spectral analysis also revealed GILT's ability to reduce cysteinylated epitopes. Such disparities in the profile of antigenic epitopes displayed by tumors and bystander APCs may contribute to tumor cell survival in the face of immunological defenses. PMID:12021307

Haque, M Azizul; Li, Ping; Jackson, Sheila K; Zarour, Hassane M; Hawes, John W; Phan, Uyen T; Maric, Maja; Cresswell, Peter; Blum, Janice S

2002-05-20

59

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

Microsoft Academic Search

Lysosomal storage diseases (LSDs) are metabolic disorders caused by enzyme deficiencies that lead to lysosomal accumulation\\u000a of undegraded substrates. Enzyme replacement therapies (ERT) have been developed as treatments for patients with Gaucher,\\u000a Niemann-Pick, Fabry, and Pompe diseases. Depending on the disease, the corresponding therapeutic enzyme is designed to be\\u000a internalized by diseased cells through receptor-mediated endocytosis via macrophage mannose receptors

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

2011-01-01

60

Selective targeting of histone methylation.  

PubMed

Histones are post-translationally modified by multiple histone-modifying enzymes, which in turn influences gene expression. Much of the work in the field to date has focused on genetic, biochemical and structural characterization of these enzymes. The most recent genome-wide methods provide insights into specific recruitment of histone-modifying enzymes in vivo and, therefore, onto mechanisms of establishing a differential expression pattern. Here we focus on the recruitment mechanisms of the enzymes involved in the placement of two contrasting histone marks, histone H3 lysine 4 (H3K4) methylation and histone H3 lysine 27 (H3K27) methylation. We describe distribution of their binding sites and show that recruitment of different histone-modifying proteins can be coordinated, opposed, or alternating. Specifically, genomic sites of the H3K4 histone demethylase KDM5A become accessible to its homolog KDM5B in cells with a lowered KDM5A level. The currently available data on recruitment of H3K4/H3K27 modifying enzymes suggests that the formed protein complexes are targeted in a sequential and temporal manner, but that additional, still unknown, interactions contribute to targeting specificity. PMID:21270517

Islam, Abul B M M K; Richter, William F; Lopez-Bigas, Nuria; Benevolenskaya, Elizaveta V

2011-02-01

61

Selective targeting of histone methylation  

PubMed Central

Histones are post-translationally modified by multiple histonemodifying enzymes, which in turn influences gene expression. Much of the work in the field to date has focused on genetic, biochemical and structural characterization of these enzymes. The most recent genome-wide methods provide insights into specific recruitment of histone-modifying enzymes in vivo and, therefore, onto mechanisms of establishing a differential expression pattern. Here we focus on the recruitment mechanisms of the enzymes involved in the placement of two contrasting histone marks, histone H3 lysine 4 (H3K4) methylation and histone H3 lysine 27 (H3K27) methylation. We describe distribution of their binding sites and show that recruitment of different histone-modifying proteins can be coordinated, opposed or alternating. Specifically, genomic sites of the H3K4 histone demethylase KDM5A become accessible to its homolog KDM5B in cells with a lowered KDM5A level. The currently available data on recruitment of H3K4/H3K27 modifying enzymes suggests that the formed protein complexes are targeted in a sequential and temporal manner, but that additional, still unknown, interactions contribute to targeting specificity.

Islam, Abul BMMK; Richter, William F; Lopez-Bigas, Nuria

2011-01-01

62

Feature Selection Applied to Radar Target Identification.  

National Technical Information Service (NTIS)

Three feature selection algorithms are investigated and applied to characterize optimum sets of frequencies for radar target identification. One algorithm is of the nonparametric discriminant analysis type, the other two algorithms, the pairwise exponenti...

O. Snorrason F. D. Garber

1987-01-01

63

In situ assay of acid sphingomyelinase and ceramidase based on LDL-mediated lysosomal targeting of ceramide-labeled sphingomyelin  

Microsoft Academic Search

The activity of lysosomal sphingolipid hydrolases is usually estimated in vitro from complex assays on cell lysates under artificial conditions including the presence of deter- gents and substrate analogs. However, the measure of their effective activity in situ (i.e., in living cells) is necessary to un- derstand the normal intracellular sphingolipid turnover. Moreover, their determination in cells from patients with

Thieny Levade; Michele Len; Denis Graber; Andre Moisand; Stephane Vermeersch; Robert Salvayre; Pierre J. Courtoyt

64

Microsatellites as targets of natural selection.  

PubMed

The ability to survey polymorphism on a genomic scale has enabled genome-wide scans for the targets of natural selection. Theory that connects patterns of genetic variation to evidence of natural selection most often assumes a diallelic locus and no recurrent mutation. Although these assumptions are suitable to selection that targets single nucleotide variants, fundamentally different types of mutation generate abundant polymorphism in genomes. Moreover, recent empirical results suggest that mutationally complex, multiallelic loci including microsatellites and copy number variants are sometimes targeted by natural selection. Given their abundance, the lack of inference methods tailored to the mutational peculiarities of these types of loci represents a notable gap in our ability to interrogate genomes for signatures of natural selection. Previous theoretical investigations of mutation-selection balance at multiallelic loci include assumptions that limit their application to inference from empirical data. Focusing on microsatellites, we assess the dynamics and population-level consequences of selection targeting mutationally complex variants. We develop general models of a multiallelic fitness surface, a realistic model of microsatellite mutation, and an efficient simulation algorithm. Using these tools, we explore mutation-selection-drift equilibrium at microsatellites and investigate the mutational history and selective regime of the microsatellite that causes Friedreich's ataxia. We characterize microsatellite selective events by their duration and cost, note similarities to sweeps from standing point variation, and conclude that it is premature to label microsatellites as ubiquitous agents of efficient adaptive change. Together, our models and simulation algorithm provide a powerful framework for statistical inference, which can be used to test the neutrality of microsatellites and other multiallelic variants. PMID:23104080

Haasl, Ryan J; Payseur, Bret A

2012-10-27

65

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

Microsoft Academic Search

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

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

1994-01-01

66

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

PubMed Central

Background The human prostacyclin receptor (hIP) undergoes agonist-induced phosphorylation, desensitisation and internalisation and may be recycled to the plasma membrane or targeted for degradation by, as yet, unknown mechanism(s). Results Herein it was sought to investigate the turnover of the hIP under basal conditions and in response to cicaprost stimulation. It was established that the hIP is subject to low-level basal degradation but, following agonist stimulation, degradation is substantially enhanced. Inhibition of the lysosomal pathway prevented basal and agonist-induced degradation of the mature species of the hIP (46-66 kDa). Conversely, inhibition of the proteasomal pathway had no effect on levels of the mature hIP but led to time-dependent accumulation of four newly synthesised immature species (38-44 kDa). It was established that both the mature and immature species of the hIP may be polyubiquitinated and this modification may be required for lysosomal sorting of the mature, internalised receptors and for degradation of the immature receptors by the 26S proteasomes through the ER-associated degradation (ERAD) process, respectively. Moreover, these data substantially advance knowledge of the factors regulating processing and maturation of the hIP, a complex receptor subject to multiple post-translational modifications including N-glycosylation, phosphorylation, isoprenylation, palmitoylation, in addition to polyubiquitination, as determined herein. Conclusion These findings indicate that the hIP is post-translationally modified by ubiquitination, which targets the immature species to the 26S proteasomal degradation pathway and the mature species to the lysosomal degradation pathway.

Donnellan, Peter D; Kinsella, B Therese

2009-01-01

67

Interaction of Salmonella enterica Serotype Typhimurium with Dendritic Cells Is Defined by Targeting to Compartments Lacking Lysosomal Membrane Glycoproteins  

PubMed Central

Dendritic cells (DCs) play a central role in the generation of acquired immunity to infections by pathogenic microorganisms. Salmonella enterica serotype Typhimurium is known to survive and proliferate intracellularly within macrophages and nonphagocytic cells, but no data exist on how this pathogen interacts with DCs. In this report, we show the capacity of serotype Typhimurium to survive within the established mouse DC line CB1. In contrast to the case for the macrophage model, the compartments of DCs containing serotype Typhimurium are devoid of lysosomal membrane glycoproteins and the PhoPQ two-component regulatory system is not essential for pathogen intracellular survival.

Garcia-Del Portillo, Francisco; Jungnitz, Heidrun; Rohde, Manfred; Guzman, Carlos A.

2000-01-01

68

Use of genomics to select antibacterial targets.  

PubMed

The problem of antibiotic resistance has eroded the usefulness of our arsenal of effective antibiotics. There is a need for new strategies to discover and develop new, effective drugs. The advent of the microbial genomics era has provided a wealth of information on a variety of microorganisms. This has allowed the identification and/or validation of a number of gene products that could serve as targets for the discovery of novel antibacterial agents. New genetic techniques and approaches have arisen in an attempt to exploit this newly available genomic data. Both random and targeted gene disruption efforts have proven effective in this process. Many of these methods would have been difficult to accomplish without DNA sequence and bioinformatics analyses. Several targets have been selected to further characterize and screen for inhibitors and one has yielded two clinical candidates. PMID:16412986

Pucci, Michael J

2006-01-18

69

Remodeling of the epitope repertoire of a candidate idiotype vaccine by targeting to lysosomal degradation in dendritic cells.  

PubMed

The generation of efficacious vaccines against self-antigens expressed in tumor cells requires breakage of tolerance, and the refocusing of immune responses toward epitopes for which tolerance may not be established. While the presentation of tumor antigens by mature dendritic cells (mDC) may surpass tolerance, broadening of the antigenic repertoire remains an issue. We report that fusion of the candidate idiotype vaccine IGKV3-20 to the Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen (EBNA)-1 inhibits degradation by the proteasome and redirects processing to the lysosome. mDCs transduced with a recombinant lentivirus encoding the chimeric idiotype efficiently primed CD4+ and CD8+ cytotoxic T-cell (CTL) responses that lysed autologous blasts expressing IGKV3-20 or pulsed with IGKV3-20 synthetic peptides, and HLA-matched IGKV3-20-positive tumor cell lines. Comparison of the cytotoxic response of CD4+ and CD8+ T lymphocytes activated by mDCs expressing the wild-type or chimeric IGKV3-20 reveled largely non-overlapping epitope repertoires in both CD4+ and CD8+ effectors. Thus, fusion to the GAr may provide an effective means to broaden the immune response to an endogenous protein by promoting the presentation of antigenic epitopes that require a lysosome-dependent processing step. PMID:22089857

Martorelli, Debora; Coppotelli, Giuseppe; Muraro, Elena; Dolcetti, Riccardo; Masucci, Maria G

2011-11-17

70

Imidazoacridinone-dependent lysosomal photodestruction: a pharmacological Trojan horse approach to eradicate multidrug-resistant cancers  

PubMed Central

Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC50 values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction.

Adar, Y; Stark, M; Bram, E E; Nowak-Sliwinska, P; van den Bergh, H; Szewczyk, G; Sarna, T; Skladanowski, A; Griffioen, A W; Assaraf, Y G

2012-01-01

71

Gamma-aminobutyric acid type B receptors are constitutively internalized via the clathrin-dependent pathway and targeted to lysosomes for degradation.  

PubMed

Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of gamma-aminobutyric acid, type B (GABAB) receptors. Therefore, we analyzed internalization of GABAB receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40-50% of cell surface receptors was detected. Stimulation of GABAB receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonist-induced internalization. The data suggest that GABAB receptors were endocytosed via the classical dynamin- and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABAB receptors via clathrin-coated pits, which resulted in lysosomal degradation of the receptors. PMID:17581821

Grampp, Thomas; Sauter, Kathrin; Markovic, Branko; Benke, Dietmar

2007-06-20

72

Target selection for the HRIBF Project  

SciTech Connect

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.

Dellwo, J. [Oak Ridge National Lab., TN (United States); Alton, G.D.; Batchelder, J.C. [Louisiana State Univ., Baton Rouge, LA (United States)

1994-12-31

73

Lysosomal disorders.  

PubMed

Although most lysosomal storage disorders present in infancy or early childhood with a progressive condition often associated with dysmorphism, considerable genetic heterogeneity exists resulting in a range of illnesses that can include a dramatic neonatal presentation. Whilst some conditions present with a characteristic neonatal phenotype (e.g. Niemann-Pick disease type C), the remainder present in a nonspecific way often with non-immune hydrops fetalis. Diagnosis can be helped by appropriate radiological studies and, in some patients, evidence of the storage phenomena can be seen in peripheral blood smears or bone marrow aspirates. Unfortunately, for the majority of affected patients no effective, curative, treatment is possible. New developments in therapy including enzyme replacement therapy and substrate deprivation may improve prognosis in some disorders. It is important to establish an accurate diagnosis, as prenatal testing can then be offered in future pregnancies. PMID:12069540

Wraith, J E

2002-02-01

74

Regionally selective changes in brain lysosomes occur in the transition from young adulthood to middle age in rats.  

PubMed

The possibility that brain aging in rats exhibits regional variations of the type found in humans was studied using lysosomal chemistry as a marker. Age-related (two vs 12months; male Sprague-Dawley) differences in cathepsin D immunostaining were pronounced in the superficial layers of entorhinal cortex and in hippocampal field CA1, but not in neocortex and field CA3. Three changes were recorded: an increase in the intraneuronal area occupied by labeled lysosomes; clumping of immunopositive material within neurons; more intense cytoplasmic staining. Western blot analyses indicated that the increases involved the active forms of cathepsin D rather than their proenzyme. Shrinkage of cathepsin-D-positive neuronal cell bodies was observed in entorhinal cortex but not in neocortical sampling zones. Age-related lysosomal changes as seen with cathepsin B immunocytochemistry were considerably more subtle than those obtained with cathepsin D antibodies. In contrast, a set of glial and/or vascular elements located in a distal dendritic field of the middle-aged hippocampus was much more immunoreactive for cathepsin B than cathepsin D. The areas exhibiting sizeable changes in the present study are reported to be particularly vulnerable to aging in humans. The results thus suggest that aspects of brain aging common to mammals help shape neurosenescence patterns in humans. PMID:10799771

Bi, X; Yong, A P; Zhou, J; Gall, C M; Lynch, G

2000-01-01

75

Effects of d -penicillamine, dichlofenac sodium and gold sodium thiomalate upon the selective release of lysosomal enzymes from human polymorphonuclear leucocytes to immune complex  

Microsoft Academic Search

The selective release of -glucuronidase (-Gluc) and -N-acetylglucosaminidase (-Glm) from human polymorphonuclear leucocytes (PMN), initiated with bovine serum albumin\\/anti-bovine serum albumin (BSA\\/anti-BSA) immune complex (15 g\\/ml–1) was significantly reduced by increasing concentrations (10–7\\u000aM, 10–6\\u000aM and 10–5\\u000aM) ofd-penicillamine (d-PEN) in a dose-dependent fashion. These effects upon the exocytosis of the lysosomal enzymes studied are in accordance with the

Olga Carevi?

1985-01-01

76

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

PubMed Central

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

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

2011-01-01

77

A Color Hierarchy for Automatic Target Selection  

PubMed Central

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

Tchernikov, Illia; Fallah, Mazyar

2010-01-01

78

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

PubMed

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

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

2013-08-06

79

Inhibition of Glioma Cell Lysosome Exocytosis Inhibits Glioma Invasion  

PubMed Central

Cancer cells invade by secreting enzymes that degrade the extracellular matrix and these are sequestered in lysosomal vesicles. In this study, the effects of the selective lysosome lysing drug GPN and the lysosome exocytosis inhibitor vacuolin-1 on lysosome exocytosis were studied to determine their effect on glioma cell migration and invasion. Both GPN and vacuolin-1 evidently inhibited migration and invasion in transwell experiments and scratch experiments. There are more lysosomes located on the cell membrane of glioma cells than of astrocytes. GPN decreased the lysosome number on the cell membrane. We found that rab27A was expressed in glioma cells, and colocalized with cathepsin D in lysosome. RNAi-Rab27A inhibited lysosome cathepsin D exocytosis and glioma cell invasion in an in vitro assay. Inhibition of cathepsin D inhibited glioma cell migration. The data suggest that the inhibition of lysosome exocytosis from glioma cells plays an important modulatory role in their migration and invasion.

Zhu, Keqing

2012-01-01

80

Inhibition of glioma cell lysosome exocytosis inhibits glioma invasion.  

PubMed

Cancer cells invade by secreting enzymes that degrade the extracellular matrix and these are sequestered in lysosomal vesicles. In this study, the effects of the selective lysosome lysing drug GPN and the lysosome exocytosis inhibitor vacuolin-1 on lysosome exocytosis were studied to determine their effect on glioma cell migration and invasion. Both GPN and vacuolin-1 evidently inhibited migration and invasion in transwell experiments and scratch experiments. There are more lysosomes located on the cell membrane of glioma cells than of astrocytes. GPN decreased the lysosome number on the cell membrane. We found that rab27A was expressed in glioma cells, and colocalized with cathepsin D in lysosome. RNAi-Rab27A inhibited lysosome cathepsin D exocytosis and glioma cell invasion in an in vitro assay. Inhibition of cathepsin D inhibited glioma cell migration. The data suggest that the inhibition of lysosome exocytosis from glioma cells plays an important modulatory role in their migration and invasion. PMID:23029308

Liu, Yu; Zhou, Yijiang; Zhu, Keqing

2012-09-28

81

Targeting of a T cell agonist peptide to lysosomes by DNA vaccination induces tolerance in the nonobese diabetic mouse  

Microsoft Academic Search

CD4 T cells are crucial effectors in the pathology of type 1 diabetes (T1D). Successful therapeutic interventions for prevention and cure of T1D in humans are still elusive. Recent research efforts have focused on the manipulation of T cells by treatment with DNA. In this paper, we studied the effects of a DNA treatment strategy designed to target antigenic peptides

E I Rivas; J P Driver; N Garabatos; M Presa; C Mora; F Rodriguez; D V Serreze; T Stratmann

2011-01-01

82

Drug induced phospholipidosis: an acquired lysosomal storage disorder.  

PubMed

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. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. PMID:22960355

Shayman, James A; Abe, Akira

2012-08-30

83

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

SciTech Connect

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.

Gonzalez-Noriega, Alfonso [Department of Cell Biology and Physiology, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, PO Box 70228, 04510 Mexico, D.F. (Mexico)]. E-mail: gonor@biomedicas.unam.mx; Ortega Cuellar, Daniel D. [Department of Cell Biology and Physiology, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, PO Box 70228, 04510 Mexico, D.F. (Mexico); Michalak, Colette [Department of Cell Biology and Physiology, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, PO Box 70228, 04510 Mexico, D.F. (Mexico)

2006-04-15

84

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

PubMed

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 NH4Cl 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. PMID:16438964

González-Noriega, Alfonso; Ortega Cuellar, Daniel D; Michalak, Colette

2006-01-24

85

Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion.  

PubMed

Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion. PMID:23337583

Zhou, Jing; Tan, Shi-Hao; Nicolas, Valérie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming

2013-01-22

86

What are the targets of photodynamic therapy?  

NASA Astrophysics Data System (ADS)

We previously classified photosensitizing agents based on sites of photodamage: mitochondria, lysosomes and the plasma membrane. More recent studies have indicated that the first target for the mitochondrial sensitizers is the anti-apoptotic protein bcl-2. Loss of bcl-2 results in a high bax/bcl-2 ratio, leading to an apoptotic response involving cytochrome c translocation. PDT with sensitizers that target lysosomes causes the release of lysosomal proteases that catalyze cleavage of the protein bid to a truncated form that can also initiate an apoptotic response. When the PDT target includes the plasma membrane, we found a greatly impaired apoptotic response, associated with caspase-3 photodamage. Sensitizers that localize in only lysosomes also catalyze variable levels of caspase photodamage, but apoptosis is only slightly impaired. PDT appears to be a highly-selective means for eradication of bcl-2, lysosomes or caspases, and can be a potentially useful tool in apoptosis research.

Kessel, David; Castelli, Michelle; Reiners, John

2002-06-01

87

Selective lysosomal uptake and accumulation of the beta-adrenergic antagonist propranolol in cultured and isolated cell systems.  

PubMed

The beta adrenoreceptor antagonist propranolol is rapidly taken up and accumulated in various cultured cell lines. When incubated in the presence of low concentrations of propranolol (10(-9) M), Hela (non-differentiated epithelia), BC3H1 (smooth muscle) and MDCK (differentiated kidney epithelia) cell cultures take up (t1/2 = 4-10 min) and accumulate the drug such that the intracellular concentration is over 1000 times that in the incubation medium. The release of propranolol from the cells was slower (t1/2 = 22 min) than the rate of uptake but the dissociation was stimulated by the addition of 1 microM propranolol to the external medium (t1/2 = 9 min). Uptake, which is non-stereoselective, is dependent on pH and is inhibited by the lysosomotropic agents, NH4Cl, methylamine and chloroquine. At higher concentrations (greater than 10(6) M), uptake is accompanied by a visual swelling of intracellular acidic vesicles staining with acridine orange. These results suggest that propranolol, a basic amphiphilic amine, is accumulated within the lysosomes of these cells. Uptake was confined to these cultured cell systems with no chloroquine-sensitive propranolol uptake, being found in isolated rabbit ventricular myocytes, red blood cells or blood platelets. Although alprenolol and cyanopindolol competed with propranolol for uptake, isoprenaline, adrenaline, noradrenaline, phenylephrine, atenolol, practolol and salbutamol were not effective inhibitors. The possible consequences of this uptake and accumulation of propranolol by certain tissues is discussed in relation to the known actions of the drug, particularly during or after abrupt withdrawal from chronic applications. PMID:3008762

Cramb, G

1986-04-15

88

Effect of target-distractor similarity on FEF visual selection in the absence of the target  

Microsoft Academic Search

We tested the hypothesis that frontal eye field (FEF) visual activity integrates visual information with a template of a target by examining whether a target that is not present in a search display influences the target selection in FEF. Neural activity was recorded in FEF of macaque monkeys performing visual search for a singleton target defined by color or direction

Takashi R. Sato; Katsumi Watanabe; Kirk G. Thompson; Jeffrey D. Schall

2003-01-01

89

Evaluating the targets of selection during character displacement.  

PubMed

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

Martin, Ryan A; Pfennig, David W

2011-06-16

90

Regulation of lamp2a levels in the lysosomal membrane.  

PubMed

The selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy depends, at least in part, on the levels of a substrate receptor at the lysosomal membrane. We have previously identified this receptor as the lysosome-associated membrane protein type 2a (lamp2a) and showed that levels of lamp2a at the lysosomal membrane directly correlate with the activity of the proteolytic pathway. Here we show that levels of lamp2a at the lysosomal membrane are mainly controlled by changes in its half-life and its distribution between the lysosomal membrane and the matrix. The lysosomal degradation of lamp2a requires the combined action of at least two different proteolytic activities at the lysosomal membrane. Lamp2a is released from the membrane by the action of these proteases, and then the truncated lamp2a is rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is a regulated process that is inhibited in the presence of substrates for chaperone-mediated autophagy and under conditions that activate that type of autophagy. Uptake of substrate proteins also results in transport of some intact lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can be reinserted back into the lysosomal membrane. The traffic of lamp2a through the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion requires the lysosomal membrane potential and protein components of the lysosomal membrane. The distribution of lamp2a between the lysosomal membrane and matrix is a dynamic process that contributes to the regulation of lysosomal membrane levels of lamp2a and consequently to the activity of the chaperone-mediated autophagic pathway. PMID:11208145

Cuervo, A M; Dice, J F

2000-07-01

91

Regulating secretory lysosomes.  

PubMed

Secretory lysosomes are lysosomes which are capable of undergoing regulated secretion in response to external stimuli. Many cells of the immune system use secretory lysosomes to release proteins involved in their specialised effector mechanisms. Precisely how lysosomal secretion is regulated in each of these cell types is now the study of much research as these mechanisms control the ability of each of these cells to function. Studies on a number of human genetic diseases have identified some key proteins in controlling secretory lysosome release, and now many interacting partners have been identified. The different regulatory components seem to vary from one cell type to another, providing a multitude of ways for fine tuning the release of secretory lysosomes. PMID:16877763

Holt, Oliver J; Gallo, Federico; Griffiths, Gillian M

2006-07-01

92

Lysosomes, cholesterol and atherosclerosis  

PubMed Central

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

Jerome, W Gray

2011-01-01

93

Functional analysis of lysosomes during mouse preimplantation embryo development.  

PubMed

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos. PMID:23080372

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Ohta, Yuki; Wada, Ayako; Ishida, Yuka; Kito, Seiji; Nishikawa, Tetsu; Minami, Naojiro; Sato, Ken; Kokubo, Toshiaki

2012-10-19

94

Myelin lesions associated with lysosomal and peroxisomal disorders.  

PubMed

Abnormalities of myelin are common in lysosomal and peroxisomal disorders. Most display a primary loss of myelin in which the myelin sheath and/or oligodendrocytes are selectively targeted by diverse pathogenetic processes. The most severe and, hence, clinically relevant are heritable diseases predominantly of infants and children, the leukodystrophies: metachromatic, globoid cell (Krabbe disease) and adreno-leukodystrophy. Our still limited understanding of these diseases has derived from multiple sources: originally, neurological-neuropathologic-neurochemical correlative studies of the natural disease in humans or other mammals, which has been enhanced by more sophisticated and contemporary techniques of cell and molecular biology. Transgenic mouse models seem to be the most promising methodology, allowing the examination of the cellular role of lysosomes and peroxisomes for formation and maintenance of both myelin and axons, and providing initial platforms to evaluate therapies. Treatment options are woefully inadequate and in their nascent stages, but still inspire some hope for the future. PMID:20819015

Faust, Phyllis L; Kaye, Edward M; Powers, James M

2010-09-01

95

Proteomics of the Lysosome  

PubMed Central

Defects in lysosomal function have been associated with numerous monogenic human diseases typically classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal proteins are also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this is the identification of its constituent proteins by proteomic analyses. To date, the mammalian lysosome has been shown to contain ~ 60 soluble luminal proteins and ~25 transmembrane proteins. However, recent proteomic studies based upon affinity purification of soluble components or subcellular fractionation to obtain both soluble and membrane components suggest that there may be many more of both classes of protein resident within this organelle than previously appreciated. Discovery of such proteins has important implications for understanding the function and the dynamics of the lysosome but can also lead the way towards the discovery of the genetic basis for human diseases of hitherto unknown etiology. Here, we describe current approaches to lysosomal proteomics and data interpretation and review the new lysosomal proteins that have recently emerged from such studies.

Lubke, Torben; Lobel, Peter; Sleat, David

2009-01-01

96

Clarifying lysosomal storage diseases  

PubMed Central

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

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

2011-01-01

97

Frequency-dependent chemolocation and chemotactic target selection  

NASA Astrophysics Data System (ADS)

Chemotaxis is typically modeled in the context of cellular motion toward a static, exogenous source of chemoattractant. Here we propose a time-dependent mechanism of chemotaxis in which a self-propelled particle (e.g. a cell) releases a chemical that diffuses to fixed particles (targets) and signals the production of a second chemical by these targets. The particle then moves up concentration gradients of this second chemical, analogous to diffusive echolocation. When one target is present, we describe probe release strategies that optimize travel of the cell to the target. In the presence of multiple targets, the one selected by the cell depends on the strength and, interestingly, on the frequency of probe chemical release. Although involving an additional chemical signaling step, our chemical 'pinging' hypothesis allows for greater flexibility in regulating target selection, as seen in a number of physical or biological realizations.

Nowak, Sarah A.; Chakrabarti, B.; Chou, Tom; Gopinathan, Ajay

2010-06-01

98

Target selection for visually-guided reaching in macaque  

PubMed Central

We examined target selection for visually-guided reaching in monkeys using a visual search task in which an odd-colored target was presented with distractors. The colors of the target and distractors were randomly switched in each trial between red and green, and the number of distractors was varied. Previous studies of saccades and attention have shown that target selection in this task is easier when a greater number of homogenous distractors is present. We found that monkeys made fewer reaches to distractors and that reaches to the target were completed more quickly when a greater number of homogenous distractors was present. When the target was presented in a sparse array of distractors, reaches had longer movement durations and greater trajectory curvature. Reaching errors were directed more often to a distractor adjacent to the target, suggesting a spatially coarse-to-fine progression during target selection. Reaches were also influenced by the properties of trials in the recent past. When the colors of the target and distractors remained the same from trial to trial rather than switching, reaches were completed more quickly and accurately, indicating that color priming across trials facilitates target selection. Moreover, when difficult search trials were randomly intermixed with easier trials without distractors, reach latencies were influenced by the difficulty of previous trials, indicating that motor initiation strategies are gradually adjusted based on accumulated experience. Overall, these results are consistent with reaching results in humans, indicating that the monkey provides a sound model for understanding the neural underpinnings of reach target selection.

Song, Joo-Hyun; Takahashi, Naomi; McPeek, Robert M.

2008-01-01

99

Automatic Target Recognition: Statistical Feature Selection of Non- Gaussian Distributed Target Classes.  

National Technical Information Service (NTIS)

Target and pattern recognition systems are in widespread use. Efforts have been made in all areas of pattern recognition to increase the performance of these systems. Feature extraction, feature selection, and classification are the major aspects of a tar...

M. J. Wilder

2011-01-01

100

Lysosomal storage disease.  

PubMed

We report a case of lysosomal storage disease diagnosed by lysosomal enzyme assay in a two year old boy with a history of gradual onset of weakness of body, poor vision, flaccid neck and spasticity in all four limbs with hyper-reflexia. On fundus examination cherry red spots were noted at macula. On performing lysosomal enzyme assay, beta-galactosidase level was considerably low. This indicates that the child is affected by lysosomal storage disease most likely GM1 gangliosidosis. The diagnosis is important because the disease is rare and it may be missed as the symptoms are similar to other neurological conditions and the diagnosis can help with future conception. PMID:20795466

Khatiwada, B; Pokharel, A

101

A selection system for identifying accessible sites in target RNAs.  

PubMed Central

Although ribozymes offer tremendous potential for posttranscriptionally controlling expression of targeted genes, their utility is often limited by the accessibility of the targeted regions within the RNA transcripts. Here we describe a method that identifies RNA regions that are accessible to oligonucleotides. Based on this selection protocol, we show that construction of hammerhead ribozymes targeted to the identified regions results in catalytic activities that are consistently and substantially greater than those of ribozymes designed on the basis of computer modeling. Identification of accessible sites should also be widely applicable to design of antisense oligonucleotides and DNAzymes.

Pan, W H; Devlin, H F; Kelley, C; Isom, H C; Clawson, G A

2001-01-01

102

The lysosomal transport of prosaposin requires the conditional interaction of its highly conserved d domain with sphingomyelin.  

PubMed

Lysosomal prosaposin (65 kDa) is a nonenzymic protein that is transported to the lysosomes in a mannose 6-phosphate-independent manner. Selective deletion of the functional domains of prosaposin indicates that the D domain and the carboxyl-terminal region are necessary for its transport to the lysosomes. Inhibitors of sphingolipid biosynthesis, such as fumonisin B(1) (FB(1)) and tricyclodecan-9-yl xanthate potassium salt (D609), also interfere with the trafficking of prosaposin to lysosomes. In this study, we examine sphingomyelin as a direct candidate for the trafficking of prosaposin. Chinese hamster ovary and COS-7 cells overexpressing prosaposin or an albumin/prosaposin construct were incubated with these inhibitors, treated with sphingolipids, and then immunostained. Sphingomyelin restored the immunostaining in lysosomes in both FB(1)- and D609-treated cells and ceramide reestablished the immunostaining in FB(1)-treated cells only. D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits glycosphingolipids, had no effect on the immunostaining pattern. To determine whether sphingomyelin has the same effect on the transport of endogenous prosaposin, testicular explants were treated with FB(1) and D609. Sphingomyelin restored prosaposin immunogold labeling in the lysosomes of FB(1)- and D609-treated Sertoli cells, whereas ceramide restored the label in FB(1) treatment only. Albumin linked to the D and COOH-terminal domains of prosaposin was used as a dominant negative competitor. The construct blocked the targeting of prosaposin and induced accumulation of membrane in the lysosomes, demonstrating that the construct uses the same transport pathway as endogenous prosaposin. In conclusion, our results showed that sphingomyelin, the D domain, and its adjacent COOH-terminal region play a crucial role in the transport of prosaposin to lysosomes. Although the precise nature of this lipid-protein interaction is not well established, it is proposed that sphingomyelin microdomains (lipid rafts) are part of a mechanism ensuring correct intercellular trafficking of prosaposin. PMID:11856752

Lefrancois, Stephane; May, Taymaa; Knight, Casey; Bourbeau, Danielle; Morales, Carlos R

2002-02-20

103

Gene Therapy for Lysosomal Storage Diseases  

Microsoft Academic Search

Lysosomal storage diseases (LSDs) comprise a diverse group of monogenetic disorders with complex clinical phenotypes that include both systemic and central nervous system pathologies. In recent years, the identification or development of mouse models recapitulating the clinical course of the LSDs has been instrumental in evaluating therapeutic strategies. Here, we review the various gene replacement strategies for target organs affected

Mark S. Sands; Beverly L. Davidson

2006-01-01

104

A Procedure for Target Market Selection in Tourism  

Microsoft Academic Search

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

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

2004-01-01

105

Target and temporal pattern selection at neocortical synapses  

Microsoft Academic Search

We attempt to summarize the properties of cortical synaptic connections and the precision with which they select their targets in the context of information processing in cortical circuits. High-frequency presyn- aptic bursts result in rapidly depressing responses at most inputs onto spiny cells and onto some interneu- rons. These 'phasic' connections detect novelty and changes in the firing rate, but

Alex M. Thomson; A. Peter Bannister; Audrey Mercer; Oliver T. Morris

2002-01-01

106

Flexible saccade-target selection in Chinese reading  

Microsoft Academic Search

As Chinese is written without orthographical word boundaries (i.e., spaces), it is unclear whether saccade targets are selected on the basis of characters or words and whether saccades are aimed at the beginning or the centre of words. Here, we report an experiment where 30 Chinese readers read 150 sentences while their eye movements were monitored. They exhibited a strong

Ming Yan; Reinhold Kliegl; Eike M. Richter; Antje Nuthmann; Hua Shu

2010-01-01

107

Selective Cell Targeting with Light-Absorbing Microparticles and Nanoparticles  

Microsoft Academic Search

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The

Costas M. Pitsillides; Edwin K. Joe; Xunbin Wei; R. Rox Anderson; Charles P. Lin

2003-01-01

108

Peptide-directed highly selective targeting of pulmonary arterial hypertension.  

PubMed

Pulmonary arterial hypertension (PAH) is a disorder of the pulmonary vasculature associated with elevated pulmonary vascular resistance. Despite recent advances in the treatment of PAH, with eight approved clinical therapies and additional therapies undergoing clinical trials, PAH remains a serious life-threatening condition. The lack of pulmonary vascular selectivity and associated systemic adverse effects of these therapies remain the main obstacles to successful treatment. Peptide-mediated drug delivery that specifically targets the vasculature of PAH lungs may offer a solution to the lack of drug selectivity. Herein, we show highly selective targeting of rat PAH lesions by a novel cyclic peptide, CARSKNKDC (CAR). Intravenous administration of CAR peptide resulted in intense accumulation of the peptide in monocrotaline-induced and SU5416/hypoxia-induced hypertensive lungs but not in healthy lungs or other organs of PAH rats. CAR homed to all layers of remodeled pulmonary arteries, ie, endothelium, neointima, medial smooth muscle, and adventitia, in the hypertensive lungs. CAR also homed to capillary vessels and accumulated in the interstitial space of the PAH lungs, manifesting its extravasation activity. These results demonstrated the remarkable ability of CAR to selectively target PAH lung vasculature and effectively penetrate and spread throughout the diseased lung tissue. These results suggest the clinical utility of CAR in the targeted delivery of therapeutic compounds and imaging probes to PAH lungs. PMID:21549345

Urakami, Takeo; Järvinen, Tero A H; Toba, Michie; Sawada, Junko; Ambalavanan, Namasivayam; Mann, David; McMurtry, Ivan; Oka, Masahiko; Ruoslahti, Erkki; Komatsu, Masanobu

2011-05-06

109

SUNS and DEBRIS surveys target selection (Phillips+, 2010)  

NASA Astrophysics Data System (ADS)

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

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

2012-01-01

110

Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation  

PubMed Central

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.

Appelqvist, Hanna; Nilsson, Cathrine; Garner, Brett; Brown, Andrew J.; Kagedal, Katarina; Ollinger, Karin

2011-01-01

111

Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation.  

PubMed

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability. PMID:21281795

Appelqvist, Hanna; Nilsson, Cathrine; Garner, Brett; Brown, Andrew J; Kågedal, Katarina; Ollinger, Karin

2011-02-01

112

Antitumor activity of metal-chelating compound Dp44mT is mediated by formation of a redox-active copper complex that accumulates in lysosomes.  

PubMed

The metal-chelating compound Dp44mT is a di-2-pyridylketone thiosemicarbazone (DpT) which displays potent and selective antitumor activity. This compound is receiving translational attention, but its mechanism is poorly understood. Here, we report that Dp44mT targets lysosome integrity through copper binding. Studies using the lysosomotropic fluorochrome acridine orange established that the copper-Dp44mT complex (Cu[Dp44mT]) disrupted lysosomes. This targeting was confirmed with pepstatin A-BODIPY FL, which showed redistribution of cathepsin D to the cytosol with ensuing cleavage of the proapoptotic BH3 protein Bid. Redox activity of Cu[Dp44mT] caused cellular depletion of glutathione, and lysosomal damage was prevented by cotreatment with the glutathione precursor N-acetylcysteine. Copper binding was essential for the potent antitumor activity of Dp44mT, as coincubation with nontoxic copper chelators markedly attenuated its cytotoxicity. Taken together, our studies show how the lysosomal apoptotic pathway can be selectively activated in cancer cells by sequestration of redox-active copper. Our findings define a novel generalized strategy to selectively target lysosome function for chemotherapeutic intervention against cancer. PMID:21750178

Lovejoy, David B; Jansson, Patric J; Brunk, Ulf T; Wong, Jacky; Ponka, Prem; Richardson, Des R

2011-07-12

113

Lysosomal storage disorders.  

PubMed

Although the first description of a lysosomal storage disorder was that of Tay-Sachs disease in 1881, the lysosome was not discovered until 1955, by Christian De Duve. The first demonstration by Hers in 1963 of a link between an enzyme deficiency and a storage disorder (Pompe's disease) paved the way for a series of seminal discoveries about the intracellular biology of these enzymes and their substrates, culminating in the successful treatment of Gaucher's disease with beta-glucosidase in the early 1990s. It is now recognized that these disorders are not simply a consequence of pure storage, but result from perturbation of complex cell signalling mechanisms. These in turn give rise to secondary structural and biochemical changes, which have important implications for therapy. Significant challenges remain, particularly the treatment of central nervous system disease. It is hoped that recent advances in our understanding of lysosomal biology will enable successful therapies to be developed. PMID:15686451

Vellodi, Ashok

2005-02-01

114

Lysosomal storage diseases  

Microsoft Academic Search

Opinion statement  \\u000a \\u000a \\u000a \\u000a \\u000a – \\u000a \\u000a •Lysosomal storage disorders (LSDs), over 40 different diseases, are now considered treatable disorders. Only a few short\\u000a years ago, Lysosomal storage disorders were seen as interesting neurodegenerative disorders without any potential for treatment.\\u000a Effective treatment strategies such as bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and glycolipid\\u000a synthesis inhibition have been developed in the last 20

Edward M. Kaye

2001-01-01

115

Hepatic Response to Lysosomal Effects of Hypoxia, Neutral Red and Chloroquine  

Microsoft Academic Search

THE histochemical detection of early changes in the characteristics, distribution and enzyme contents of lysosomes, as well as their cycles of appearance and disappearance, affords a sensitive index of toxic effects on the cell. Hypoxia is known to labilize lysosomes1 and might be expected to render them more susceptible to adverse influences. Selective uptake of neutral red by lysosomes2,3 and

R. Abraham; L. Golberg; P. Grasso

1967-01-01

116

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

PubMed

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

Casida, John E; Durkin, Kathleen A

2013-01-01

117

In vivo selection of tumor-targeting RNA motifs  

PubMed Central

In an effort to target the in vivo context of tumor-specific moieties, a large library of nuclease-resistant RNA oligonucleotides was screened in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to protein p68, an RNA helicase that has been shown to be upregulated in colorectal cancer.

Mi, Jing; Liu, Yingmiao; Rabbani, Zahid N.; Yang, Zhongguang; Urban, Johannes H; Sullenger, Bruce A.; Clary, Bryan M.

2009-01-01

118

In vivo selection of tumor-targeting RNA motifs.  

PubMed

In an effort to target the in vivo context of tumor-specific moieties, we screened a large library of nuclease-resistant RNA oligonucleotides in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to p68, an RNA helicase that has been shown to be upregulated in colorectal cancer. PMID:19946274

Mi, Jing; Liu, Yingmiao; Rabbani, Zahid N; Yang, Zhongguang; Urban, Johannes H; Sullenger, Bruce A; Clary, Bryan M

2009-11-29

119

Lysosomal lipid storage diseases.  

PubMed

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

Schulze, Heike; Sandhoff, Konrad

2011-06-01

120

A negative selection heuristic to predict new transcriptional targets  

PubMed Central

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.

2013-01-01

121

Nigericin selectively targets cancer stem cells in nasopharyngeal carcinoma.  

PubMed

Nasopharyngeal carcinoma (NPC) is prevalent in southern China, northern Africa, and Alaska. The prognosis for NPC patients at early stage is good, while it is poor for patients at late stages. Cancer stem cells (CSCs) have been proposed to be associated with tumor initiation, relapse and metastasis, and the poor prognosis of NPC likely results from residual CSCs after therapy. Study on the therapy targeting CSCs in NPC remains poor, though it received intensive attentions in other cancers. Here, we used NPC cell lines with high and low proportion of CSCs as models to explore the effect of nigericin, an antibiotic, on CSCs. We found that nigericin could selectively target CSCs and sensitize CSCs in NPC to the widely used clinical drug cisplatin both in vitro and in vivo. Moreover, downregulation of the polycomb group protein Bmi-1 may contribute to the inhibitory effect of nigericin on CSCs. Furthermore, by using the in vitro NPC cell models, we found that nigericin could significantly decrease the migration and invasion abilities, which are known to be associated with CSCs. Taken together, our results suggest that nigericin can selectively target CSCs in NPC, which could be a candidate CSCs targeting drug for clinical evaluation. PMID:23831840

Deng, Cheng-Cheng; Liang, Yi; Wu, Man-Si; Feng, Fu-Tuo; Hu, Wen-Rong; Chen, Li-Zhen; Feng, Qi-Sheng; Bei, Jin-Xin; Zeng, Yi-Xin

2013-07-04

122

Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology  

NSDL National Science Digital Library

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

Emma J. Parkinson-Lawrence (South Australian Pathology Services); Tetyana Shandala (South Australian Pathology Services); Mark Prodoehl (South Australian Pathology Services); Revecca Plew (South Australian Pathology Services); Glenn N. Borlace (South Australian Pathology Services); Doug A. Brooks (South Australian Pathology Services)

2010-04-01

123

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

PubMed Central

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

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

2013-01-01

124

Think Outside the Color Box: Probabilistic Target Selection and the SDSS-XDQSO Quasar Targeting Catalog  

NASA Astrophysics Data System (ADS)

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 <~ z <~ 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 <= z <= 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 deg2 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.

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

2011-03-01

125

SELECTION, PRIORITIZATION, AND CHARACTERISTICS OF KEPLER TARGET STARS  

SciTech Connect

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.

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

2010-04-20

126

Selective Inhibition of Retinal Angiogenesis by Targeting PI3 Kinase  

PubMed Central

Ocular neovascularisation is a pathological hallmark of some forms of debilitating blindness including diabetic retinopathy, age related macular degeneration and retinopathy of prematurity. Current therapies for delaying unwanted ocular angiogenesis include laser surgery or molecular inhibition of the pro-angiogenic factor VEGF. However, targeting of angiogenic pathways other than, or in combination to VEGF, may lead to more effective and safer inhibitors of intraocular angiogenesis. In a small chemical screen using zebrafish, we identify LY294002 as an effective and selective inhibitor of both developmental and ectopic hyaloid angiogenesis in the eye. LY294002, a PI3 kinase inhibitor, exerts its anti-angiogenic effect in a dose-dependent manner, without perturbing existing vessels. Significantly, LY294002 delivered by intraocular injection, significantly inhibits ocular angiogenesis without systemic side-effects and without diminishing visual function. Thus, targeting of PI3 kinase pathways has the potential to effectively and safely treat neovascularisation in eye disease.

Alvarez, Yolanda; Astudillo, Olaya; Jensen, Lasse; Reynolds, Alison L.; Waghorne, Nora; Brazil, Derek P.; Cao, Yihai; O'Connor, John J.; Kennedy, Breandan N.

2009-01-01

127

Lysosomal Storage Diseases  

Microsoft Academic Search

\\u000a The lysosomal storage diseases (LSD) are a heterogeneous group of disorders, characterized by the progressive accumulation\\u000a of various substrates in multiple cell types, as a consequence of defects in the degradation of by-products of cellular turnover.\\u000a Several subtypes are associated with neurodegenerative features, which present as a major therapeutic challenge. Although\\u000a the causal gene defects and corresponding enzyme, cofactor, or

Gregory M. Pastores

128

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

PubMed

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

Lamore, Sarah D; Wondrak, Georg T

2011-07-20

129

Newborn screening for lysosomal storage disorders.  

PubMed

Lysosomes are intracellular organelles containing acid hydrolases that degrade biological macromolecules. Lysosomal storage disorders (LSDs) are caused by absent activity of one or more of these enzymes due to mutations of genes encoding lysosomal hydrolases or enzymes that process, target, and transport these enzymes. The specific signs and symptoms of each LSD derive from the type of material accumulated within the lysosome, the site (organ) of accumulation and the response of the body (sometimes in the form of an inflammatory or immune response) to the accumulated material. Interest for inclusion of these disorders in newborn screening programs derives from the availability of effective therapy in the form of enzyme replacement or substrate reduction therapy and bone marrow transplant that may improve long-term outcome especially if started prior to irreversible organ damage. Based on the availability of therapy and suitable screening methods, Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidosis I and II, Niemann-Pick disease, and Krabbe disease are candidates for newborn screening. Pilot newborn screening projects have been performed for some of these conditions that indicate the feasibility of this approach. This review will provide insight into these screening strategies and discuss their advantages and limitations. © 2011 Wiley-Liss, Inc. PMID:21312327

Nakamura, Kimitoshi; Hattori, Kiyoko; Endo, Fumio

2011-02-10

130

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

NSDL National Science Digital Library

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

Emma J. Parkinson-Lawrence (South Australian Pathology Services); Tetyana Shandala (South Australian Pathology Services); Mark Prodoehl (South Australian Pathology Services); Revecca Plew (South Australian Pathology Services); Glenn N. Borlace (South Australian Pathology Services); Doug A. Brooks (South Australian Pathology Services)

2010-04-01

131

Lysosomal protease expression in mature enamel.  

PubMed

The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix. PMID:18703868

Tye, Coralee E; Lorenz, Rachel L; Bartlett, John D

2008-08-15

132

The cell biology of lysosomal storage disorders  

Microsoft Academic Search

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

Anthony H. Futerman; Gerrit van Meer

2004-01-01

133

Endocannabinoids Prevent ?-Amyloid-mediated Lysosomal Destabilization in Cultured Neurons*  

PubMed Central

Neuronal cell loss underlies the pathological decline in cognition and memory associated with Alzheimer disease (AD). Recently, targeting the endocannabinoid system in AD has emerged as a promising new approach to treatment. Studies have identified neuroprotective roles for endocannabinoids against key pathological events in the AD brain, including cell death by apoptosis. Elucidation of the apoptotic pathway evoked by ?-amyloid (A?) is thus important for the development of therapeutic strategies that can thwart A? toxicity and preserve cell viability. We have previously reported that lysosomal membrane permeabilization plays a distinct role in the apoptotic pathway initiated by A?. In the present study, we provide evidence that the endocannabinoid system can stabilize lysosomes against A?-induced permeabilization and in turn sustain cell survival. We report that endocannabinoids stabilize lysosomes by preventing the A?-induced up-regulation of the tumor suppressor protein, p53, and its interaction with the lysosomal membrane. We also provide evidence that intracellular cannabinoid type 1 receptors play a role in stabilizing lysosomes against A? toxicity and thus highlight the functionality of these receptors. Given the deleterious effect of lysosomal membrane permeabilization on cell viability, stabilization of lysosomes with endocannabinoids may represent a novel mechanism by which these lipid modulators confer neuroprotection.

Noonan, Janis; Tanveer, Riffat; Klompas, Allan; Gowran, Aoife; McKiernan, Joanne; Campbell, Veronica A.

2010-01-01

134

Selecting appropriate test targets for portal weapons detection  

SciTech Connect

Adjusting a portal metal detector for weapons detection must be performed with great care. Set the sensitivity too high and the false rejection rate for personnel passing through the portal will slow traffic to everyone's annoyance. Set the sensitivity too low and run the risk that the machine will allow some of today's small handguns to pass with impunity. Because many of the modern handguns are so small the difference between a sensitivity setting that is too high and one that is too low is quite small. In order to find that fine line between too much sensitivity and too little, a set of test targets must be selected that represent the widest possible spectrum of weapons. The test set must not only be representative of small guns but also reflect the wide variety of metals that are used to manufacture today's weapons.

Murray, D.W.

1990-01-01

135

Subtype-selective targeting of voltage-gated sodium channels  

PubMed Central

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.

England, Steve; de Groot, Marcel J

2009-01-01

136

TM7SF1 (GPR137B): a novel lysosome integral membrane protein.  

PubMed

In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane. PMID:22729905

Gao, Jialin; Xia, Libin; Lu, Meiqing; Zhang, Binhua; Chen, Yueping; Xu, Rang; Wang, Lizhuo

2012-06-24

137

Selecting analytical target pesticides in monitoring: Sensitivity analysis and scoring.  

PubMed

Measuring river water concentrations of all pesticides applied in a catchment area is a daunting task. This study aims to develop new score tables for selecting analytical target pesticides. Sensitivity analyses were conducted using a diffuse pollution hydrologic model to quantitatively evaluate the influence of pesticide properties (e.g., log K(OC), degradability [half-life]) on concentrations of rice-farming pesticides in river water. Using the results of the analyses, score tables were systematically designed for the pesticide properties such that the sum of the scores for a particular pesticide, designated as the contamination index, was proportional to the expected/predicted concentration of that pesticide in river water. The contamination indexes for pesticides applied in three river basins were calculated and compared with the corresponding observed pesticide concentrations. Correlations between contamination indexes and observed concentrations were fairly good. Pesticides were ranked according to the quotients obtained by dividing the pesticide concentrations predicted from the contamination indexes by the corresponding drinking-water quality guideline values, and pesticide candidates to be monitored were successfully selected on the basis of a threshold quotient. PMID:22154284

Tani, Koji; Matsui, Yoshihiko; Iwao, Kensuke; Kamata, Motoyuki; Matsushita, Taku

2011-11-25

138

Regulated secretion of conventional lysosomes  

Microsoft Academic Search

Regulated secretion has been traditionally regarded as a specialized process present in only a few cell types. Similarly, the secretory lysosomes of hematopoietic cells have been viewed as ‘modified’ organelles that acquired the machinery for regulated exocytosis. However, there is evidence that conventional lysosomes can, in many cell types, respond to rises in the intracellular free Ca2+ concentration by fusing

Norma W Andrews

2000-01-01

139

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

PubMed

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

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

2011-09-29

140

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

PubMed Central

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.

Str?mme, 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

141

The role of lysosomes in limiting drug toxicity in mice.  

PubMed

The distribution behavior of a drug within a cell is an important, yet often overlooked, variable in both activity and differential selectivity. In normal cells, drugs with weakly basic properties are known to be extensively compartmentalized in acidic organelles such as lysosomes via ion trapping. Several cancer cell lines have been shown to have defective acidification of endocytic organelles and therefore have a diminished capacity to sequester such lysosomotropic agents. In this study, we tested the hypothesis that the low lysosomal pH of normal cells plays an important role in protecting normal tissues from the toxic effects of lysosomotropic anticancer drugs. The influence of lysosomal pH status on the toxicity of inhibitors of the molecular chaperone Hsp90 that did or did not possess lysosomotropic properties was evaluated in mice. Toxicity of Hsp90 inhibitors was evaluated in normal mice and in mice treated with chloroquine to elevate lysosomal pH by assessing morbidity and utilizing biochemical assays to diagnose hepatic and renal toxicity. Toxicity of the lysosomotropic inhibitor 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) was significantly enhanced in mice with elevated lysosomal pH relative to mice with normal lysosomal pH. In contrast, elevation of lysosomal pH had no significant impact on toxicity of the nonlysosomotropic inhibitor geldanamycin. These results support the notion that the low lysosomal pH of normal cells plays an important role in protecting these cells from the toxic effects of anticancer agents with lysosomotropic properties and has implications for the design/selection of anticancer drugs with improved safety and differential selectivity. PMID:20056778

Ndolo, Rosemary A; Forrest, M Laird; Krise, Jeffrey P

2010-01-07

142

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

PubMed

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

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

2010-03-15

143

Autophagy in lysosomal storage disorders  

PubMed Central

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

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

2012-01-01

144

Consideration of Molecular Weight during Compound Selection in Virtual Target-Based Database Screening  

Microsoft Academic Search

Virtual database screening allows for millions of chemical compounds to be computationally selected based on structural complimentarity to known inhibitors or to a target binding site on a biological macromolecule. Compound selection in virtual database screening when targeting a biological macromolecule is typically based on the interaction energy between the chemical compound and the target macromolecule. In the present study

Yongping Pan; Niu Huang; Sam Cho; Alexander D. MacKerell Jr.

2003-01-01

145

Mice deficient in lysosomal acid phosphatase develop lysosomal storage in the kidney and central nervous system.  

PubMed

Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression. Neither the physiological substrates nor the functional significance is known. Mice with a deficiency of LAP generated by targeted disruption of the LAP gene are fertile and develop normally. Microscopic examination of various peripheral organs revealed progredient lysosomal storage in podocytes and tubular epithelial cells of the kidney, with regionally different ultrastructural appearance of the stored material. Within the central nervous system, lysosomal storage was detected to a regionally different extent in microglia, ependymal cells, and astroglia concomitant with the development of a progressive astrogliosis and microglial activation. Whereas behavioral and neuromotor analyses were unable to distinguish between control and deficient mice, approximately 7% of the deficient animals developed generalized seizures. From the age of 6 months onward, conspicuous alterations of bone structure became apparent, resulting in a kyphoscoliotic malformation of the lower thoracic vertebral column. We conclude from these findings that LAP has a unique function in only a subset of cells, where its deficiency causes the storage of a heterogeneously appearing material in lysosomes. The causal relationship of the enzyme defect to the clinical manifestations remains to be determined. PMID:9228031

Saftig, P; Hartmann, D; Lüllmann-Rauch, R; Wolff, J; Evers, M; Köster, A; Hetman, M; von Figura, K; Peters, C

1997-07-25

146

Neospora caninum: infection induces high lysosomal activity.  

PubMed

Neospora caninum is a protozoan that causes abortion in cattle and neuromuscular lesions in dogs, making it an important target of veterinary medicine. Lysosomes are cellular organelles responsible for important biological functions as cellular defense mechanisms. The aim of this work was to evaluate the lysosomal stability of rat gliocytes infected in vitro with N. caninum. Rat glial cultures were infected at a ratio of 1:1 (cell/parasite). The enzymatic activity of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was assayed in the medium of control and infected cell cultures. The activity observed at 24h of incubation was 0.4±0.08mU/mg/min for control cells and 1.3±0.5mU/mg/min for infected cells. After 72h, control and infected cells exhibited activities of 1.3±0.5 and 4.1±0.9mU/mg/min, respectively. These results suggested that lysosomal compartment plays an important role in the mechanisms of cellular infection by N. caninum. PMID:23648665

Pinheiro, Alexandre M; Santos, Cláudia Valle Cabral D; Rodrigues, Luiz Erlon A

2013-05-03

147

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

NSDL National Science Digital Library

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

Emma J. Parkinson-Lawrence (South Australian Pathology Services); Tetyana Shandala (South Australian Pathology Services); Mark Prodoehl (South Australian Pathology Services); Revecca Plew (South Australian Pathology Services); Glenn N. Borlace (South Australian Pathology Services); Doug A. Brooks (South Australian Pathology Services)

2010-04-01

148

Lysosomal cell death at a glance.  

PubMed

Lysosomes serve as the cellular recycling centre and are filled with numerous hydrolases that can degrade most cellular macromolecules. Lysosomal membrane permeabilization and the consequent leakage of the lysosomal content into the cytosol leads to so-called "lysosomal cell death". This form of cell death is mainly carried out by the lysosomal cathepsin proteases and can have necrotic, apoptotic or apoptosis-like features depending on the extent of the leakage and the cellular context. This article summarizes our current knowledge on lysosomal cell death with an emphasis on the upstream mechanisms that lead to lysosomal membrane permeabilization. PMID:23720375

Aits, Sonja; Jäättelä, Marja

2013-05-01

149

Transmembrane Domain Mutations Influence the Cellular Distribution of Lysosomal Membrane Glycoprotein A  

Microsoft Academic Search

The lgp\\/LAMP family of mammalian and avian lysosomal type I membrane glycoproteins features short, conserved, cytosolic tails that possess lysosomal targeting information. The sequences of the adjacent transmembrane domains are also highly conserved, with six amino acids identical in all sixteen known lgp variants. These six residues are found along one side of a hypothetical alpha-helix that may comprise this

Susan Wimer-Mackin; Bruce L. Granger

1996-01-01

150

Distinct Mechanisms of Ferritin Delivery to Lysosomes in Iron-Depleted and Iron-Replete Cells ?  

PubMed Central

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation.

Asano, Takeshi; Komatsu, Masaaki; Yamaguchi-Iwai, Yuko; Ishikawa, Fuyuki; Mizushima, Noboru; Iwai, Kazuhiro

2011-01-01

151

Lysosomal trafficking functions of mucolipin-1 in murine macrophages  

PubMed Central

Background Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes. Results We show that mucolipin-1 localizes to lysosomal compartments in RAW264.7 mouse macrophages that show subcompartmental accumulations of endocytosed molecules. Using stable RNAi clones, we show that mucolipin-1 is required for the exit of lipids from these compartments, for the transport of endocytosed molecules to terminal lysosomes, and for the transport of the Major Histocompatibility Complex II to the plasma membrane. Conclusion Mucolipin-1 functions in the efficient exit of molecules, destined for various cellular organelles, from lysosomal compartments.

Thompson, Eric G; Schaheen, Lara; Dang, Hope; Fares, Hanna

2007-01-01

152

Lysosomal photodamage induces cell death via mitochondrial apoptotic pathway  

NASA Astrophysics Data System (ADS)

Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using real-time single-cell analysis. In this study, the fluorescence probes Cyto c-GFP and DsRed-Mit were used to detect the spatial and temporal changes of cytochrome c in real-time in sub-cell level; the Rhodamine 123 dyes were used to monitor the changes of mitochondrial membrane potential. The results showed that, after PDT treatment,the mitochondrial membrane potential decreased, and cytochrome c released from mitochondria; The caspase-3 was activated obviously. These results suggested that lysosomal photodamage activates mitochondrial apoptotic pathway to induce cell death.

Liu, Lei; Wang, Xian-Wang; Li, Hui

2009-11-01

153

syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes.  

PubMed Central

B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated gamma-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated gamma-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of gamma-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for gamma-chain-mediated FcR delivery to lysosomes.

Bonnerot, C; Briken, V; Brachet, V; Lankar, D; Cassard, S; Jabri, B; Amigorena, S

1998-01-01

154

Epidermal Growth Factor Receptor-Targeted Photosensitizer Selectively Inhibits EGFR Signaling and Induces Targeted Phototoxicity In Ovarian Cancer Cells  

PubMed Central

Targeted photosensitizer delivery to EGFR expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyin monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody.

Abu-Yousif, Adnan O.; Moor, Anne C. E.; Zheng, Xiang; Savellano, Mark D.; Yu, Weiping; Selbo, Pal K.; Hasan, Tayyaba

2012-01-01

155

Fading Characteristics of Panchromatic Radar Backscatter from Selected Agricultural Targets  

Microsoft Academic Search

An experiment was performed to determine the fading characteristics of backscattered radar signals from four agricultural targets at 9 GHz. The targets included two different row crops (corn and soybeans), a continuous canopy (alfalfa) and bare ground. After a short review of the statistics of Rayleigh fading backscatter, the data processing method and the results of the experiment are analyzed.

Thomas F. Bush; Fawwaz Uloby

1975-01-01

156

Saccade target selection: Do distractors affect saccade accuracy?  

Microsoft Academic Search

A study is reported in which eye movements were recorded when observers attempted to make a saccade to a target in the presence of a nearby and visually identical distractor. It was found that saccade targeting accuracy was completely unaffected by the presence of the distractor, except in the cases where the dis- tractor was on the same axis as

John M. Findlay; Hazel I. Blythe

2008-01-01

157

Predicting selective drug targets in cancer through metabolic networks  

Microsoft Academic Search

The interest in studying metabolic alterations in cancer and their potential role as novel targets for therapy has been rejuvenated in recent years. Here, we report the development of the first genome-scale network model of cancer metabolism, validated by correctly identifying genes essential for cellular proliferation in cancer cell lines. The model predicts 52 cytostatic drug targets, of which 40%

Ori Folger; Livnat Jerby; Christian Frezza; Eyal Gottlieb; Eytan Ruppin; Tomer Shlomi

2011-01-01

158

Fading characteristics of panchromatic radar backscatter from selected agricultural targets  

Microsoft Academic Search

An experiment was performed to empirically determine the fading characteristics of backscattered radar signals from four agricultural targets at 9 GHz. After a short review of the statistics of Rayleigh fading backscatter, the data processing method and results of the data are analyzed. Comparison with theory shows adequate agreement with the experimental results, provided of course, the targets are modeled

T. F. Bush; F. T. Ulaby

1973-01-01

159

Reversal of a distractor effect on saccade target selection after superior colliculus inactivation  

PubMed Central

Recent evidence indicates that inactivation of the primate superior colliculus (SC) results in an increase in saccade target selection errors. The pattern of errors suggests that a winner-take-all competition selects the saccade goal, and that SC inactivation perturbs this process by biasing the competition against stimuli in the inactivated field. To investigate this idea, the difficulty of target selection was manipulated in a color-oddity search task by varying the number of homogenous distractors in the search array. Previous studies have shown that target selection is easier when a greater number of homogenous distractors is present, due to perceptual grouping of the distractors. These results were replicated when testing with the SC intact. Surprisingly, during SC inactivation, this normal trend was reversed: target selection performance declined significantly with more distractors, resulting in a greater proportion of errant saccades to distractors. Examination of the saccade endpoints indicates that after SC inactivation, many errant saccades were directed to distractors adjacent to the target. This pattern of results suggests that the salience signal used by the SC for target selection is relatively broad in spatial scope. As a result, when the area of the SC representing the target location is inactivated, distractors near the target are at a competitive advantage relative to more distant distractors, and consequently, are selected more often as the saccade goal. This contributes to the trend of worse performance with more distractors due to the greater proximity of distractors to the target.

McPeek, Robert M.

2008-01-01

160

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

ERIC Educational Resources Information Center

|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…

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

2011-01-01

161

Selection of antisense oligonucleotides based on multiple predicted target mRNA structures  

Microsoft Academic Search

Background: Local structures of target mRNAs play a significant role in determining the efficacies of antisense oligonucleotides (ODNs), but some structure-based target site selection methods are limited by uncertainties in RNA secondary structure prediction. If all the predicted structures of a given mRNA within a certain energy limit could be used simultaneously, target site selection would obviously be improved in

Xiaochen Bo; Shaoke Lou; Daochun Sun; Wenjie Shu; Jing Yang; Shengqi Wang

2006-01-01

162

Lysosome-mediated processing of chromatin in senescence.  

PubMed

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

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

2013-07-01

163

Target track extraction in high false density environments using multiple hypothetical frame selection MLPDA  

NASA Astrophysics Data System (ADS)

MLPDA (Maximum Likelihood Probabilistic Data Association) has drawn attention as an effective target track extraction algorithm in high false density environments. In this algorithm, the target track is estimated as the maximum likelihood state vector, by using multiple observation frames that include the target signal and many false signals. The track is confirmed whether it is the true target or not, by comparing its likelihood with a given track confirmation threshold. However, when the target signals are lost at several frames, the conventional MLPDA deteriorates the track estimation accuracy due to false signals in frames without the target signal. In this paper, we propose multiple hypothetical frame selection MLPDA, which can extract the target track under the situation where the target signals are lost in several frames. Specifically, a batch of stored frames is first selected for track extraction. If the track is not confirmed, our algorithm offers multiple frame selection hypotheses where some frames are assumed to be the frames without the target signal and the other frames include the target signal. The track is extracted under these hypotheses, respectively, and the most likely hypothesis is accepted. If all hypotheses are rejected, our proposed method generates hypotheses that increase the number of frames without the target signal, and verifies them again. Furthermore, the hypotheses that have likelihoods above a given threshold are retained in order to modify the wrong frame selection later. Simulation results show the validity of our proposed method.

Mori, Masanori; Matsuzaki, Takashi; Kameda, Hiroshi; Umezawa, Toru

2013-09-01

164

Eye-Hand Coordination During Target Selection in a Pop-Out Visual Search  

PubMed Central

We examined the coordination of saccades and reaches in a visual search task in which monkeys were rewarded for reaching to an odd-colored target among distractors. Eye movements were unconstrained, and monkeys typically made one or more saccades before initiating a reach. Target selection for reaching and saccades was highly correlated with the hand and eyes landing near the same final stimulus both for correct reaches to the target and for incorrect reaches to a distractor. Incorrect reaches showed a bias in target selection: they were directed to the distractor in the same hemifield as the target more often than to other distractors. A similar bias was seen in target selection for the initial saccade in correct reaching trials with multiple saccades. We also examined the temporal coupling of saccades and reaches. In trials with a single saccade, a reaching movement was made after a fairly stereotyped delay. In multiple-saccade trials, a reach to the target could be initiated near or even before the onset of the final target-directed saccade. In these trials, the initial trajectory of the reach was often directed toward the fixated distractor before veering toward the target around the time of the final saccade. In virtually all cases, the eyes arrived at the target before the hand, and remained fixated until reach completion. Overall, these results are consistent with flexible temporal coupling of saccade and reach initiation, but fairly tight coupling of target selection for the two types of action.

McPeek, Robert M.

2009-01-01

165

Microbial metabolomics: replacing trial-and-error by the unbiased selection and ranking of targets  

Microsoft Academic Search

Microbial production strains are currently improved using a combination of random and targeted approaches. In the case of a targeted approach, potential bottlenecks, feed-back inhibition, and side-routes are removed, and other processes of interest are targeted by overexpressing or knocking-out the gene(s) of interest. To date, the selection of these targets has been based at its best on expert knowledge,

Mariët J. van der Werf; Renger H. Jellema; Thomas Hankemeier

2005-01-01

166

Signaling from lysosomes to mitochondria sensitizes cancer cells to photodynamic treatment  

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

167

Genome-wide polymorphisms show unexpected targets of natural selection  

PubMed Central

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

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

2012-01-01

168

Tumour selective targeting of cell cycle kinases for cancer treatment.  

PubMed

The deregulation of the cell cycle and checkpoint machinery in cancer presents a highly attractive therapeutic strategy. We review here the strategies used to exploit the dysregulated cell cycle, both through targeting kinases required for cell cycle progression, and checkpoint kinases to inappropriately force cells through the cell cycle. Appropriate control of the cell cycle is critical for proliferating normal cells, and we discuss the importance of defining tumour specific vulnerabilities that could be targeted with cell cycle kinase inhibitors. Recent studies have shown that ER-positive breast cancers rely on CDK4 to promote proliferation. TP53 mutant cancer cell lines are sensitive to WEE1 and CHK1 inhibitors in combination with chemotherapy, while PTEN-deficient aneuploid cancer cell lines are sensitive to TTK inhibitors. PMID:23597425

Aarts, Marieke; Linardopoulos, Spiros; Turner, Nicholas C

2013-04-15

169

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

PubMed

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

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

2013-07-02

170

The unfolded protein response selectively targets active smoothened mutants.  

PubMed

The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

2013-04-09

171

Selection of antisense oligonucleotides based on multiple predicted target mRNA structures  

PubMed Central

Background Local structures of target mRNAs play a significant role in determining the efficacies of antisense oligonucleotides (ODNs), but some structure-based target site selection methods are limited by uncertainties in RNA secondary structure prediction. If all the predicted structures of a given mRNA within a certain energy limit could be used simultaneously, target site selection would obviously be improved in both reliability and efficiency. In this study, some key problems in ODN target selection on the basis of multiple predicted target mRNA structures are systematically discussed. Results Two methods were considered for merging topologically different RNA structures into integrated representations. Several parameters were derived to characterize local target site structures. Statistical analysis on a dataset with 448 ODNs against 28 different mRNAs revealed 9 features quantitatively associated with efficacy. Features of structural consistency seemed to be more highly correlated with efficacy than indices of the proportion of bases in single-stranded or double-stranded regions. The local structures of the target site 5' and 3' termini were also shown to be important in target selection. Neural network efficacy predictors using these features, defined on integrated structures as inputs, performed well in "minus-one-gene" cross-validation experiments. Conclusion Topologically different target mRNA structures can be merged into integrated representations and then used in computer-aided ODN design. The results of this paper imply that some features characterizing multiple predicted target site structures can be used to predict ODN efficacy.

Bo, Xiaochen; Lou, Shaoke; Sun, Daochun; Shu, Wenjie; Yang, Jing; Wang, Shengqi

2006-01-01

172

Preventing lysosomal fat indigestion.  

PubMed

Autophagy contributes to lipid catabolism through direct mobilization and breakdown of cellular lipid stores. Two recent studies reveal the regulatory mechanisms activated by cells during starvation to ensure that the cellular compartments involved in autophagic lipid catabolism are ready to receive, process and use these lipids. The regulators represent attractive therapeutic targets to help fight lipid-excess-associated diseases. PMID:23728462

Cuervo, Ana Maria

2013-06-01

173

Comparison of basis-vector selection methods for target and background subspaces as applied to subpixel target detection  

NASA Astrophysics Data System (ADS)

This paper focuses on comparing three basis-vector selection techniques as applied to target detection in hyperspectral imagery. The basis-vector selection methods tested were the singular value decomposition (SVD), pixel purity index (PPI), and a newly developed approach called the maximum distance (MaxD) method. Target spaces were created using an illumination invariant technique, while the background space was generated from AVIRIS hyperspectral imagery. All three selection techniques were applied (in various combinations) to target as well as background spaces so as to generate dimensionally-reduced subspaces. Both target and background subspaces were described by linear subspace models (i.e., structured models). Generated basis vectors were then implemented in a generalized likelihood ratio (GLR) detector. False alarm rates (FAR) were tabulated along with a new summary metric called the average false alarm rate (AFAR). Some additional summary metrics are also introduced. Impact of the number of basis vectors in the target and background subspaces on detector performance was also investigated. For the given AVIRIS data set, the MaxD method as applied to the background subspace outperformed the other two methods tested (SVD and PPI).

Bajorski, Peter; Ientilucci, Emmett J.; Schott, John R.

2004-08-01

174

Targeting Nucleic Acid Secondary Structures by Antisense Oligonucleotides Designed through in vitro Selection  

NASA Astrophysics Data System (ADS)

Using an in vitro selection approach, we have isolated oligonucleotides that can bind to a DNA hairpin structure. Complex formation of these oligonucleotides with the target hairpin involves some type of triple-stranded structure with noncanonical interaction, as indicated by band-shift assays and footprinting studies. The selected oligomers can block restriction endonuclease cleavage of the target hairpin in a sequence-specific manner. We demonstrate that in vitro selection can extend the antisense approach to functional targeting of secondary structure motifs. This could provide a basis for interfering with regulatory processes mediated by a variety of nucleic acid structures.

Mishra, Rakesh K.; Le Tinevez, Rejane; Toulme, Jean-Jacques

1996-10-01

175

Lysosomal Cathepsin B: Correlation with Metastatic Potential  

Microsoft Academic Search

Although lysosomal enzymes are implicated in the processes of tumor invasion and metastasis, their cellular origin within the tumor is unclear. The activity of the lysosomal proteinase cathepsin B is significantly elevated in a variant of the B16 melanoma with high metastatic potential. The cathepsin B activity is localized to the lysosomes of the tumor cells.

Bonnie F. Sloane; John R. Dunn; Kenneth V. Honn

1981-01-01

176

Lysosomal Cathepsin B: Correlation with Metastatic Potential  

NASA Astrophysics Data System (ADS)

Although lysosomal enzymes are implicated in the processes of tumor invasion and metastasis, their cellular origin within the tumor is unclear. The activity of the lysosomal proteinase cathepsin B is significantly elevated in a variant of the B16 melanoma with high metastatic potential. The cathepsin B activity is localized to the lysosomes of the tumor cells.

Sloane, Bonnie F.; Dunn, John R.; Honn, Kenneth V.

1981-06-01

177

Targeted Selection of Recombinant Clones through Gene Dosage Effects  

Microsoft Academic Search

The availability of multicopy plasmid vectors for the yeast Saccharomyces cerevisiae allows the selective amplification of individual segments of the genome. Increased dosage of particular genes results in overproduction of specific gene products and thereby confers resistance to certain metabolic inhibitors. Advantage was taken of this fact to isolate recombinant clones that increase the activities of the enzymes UDP-N-acetylglucosamine-1-P transferase

Jasper Rine; William Hansen; Edna Hardeman; Ronald W. Davis

1983-01-01

178

Immunochemistry of Lysosomal Storage Disorders  

Microsoft Academic Search

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

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

2006-01-01

179

Patient selection for ventricular assist devices: a moving target.  

PubMed

The number of patients with advanced heart failure that has become unresponsive to conventional medical therapy is increasing rapidly. One of the most promising new alternatives to heart transplantation is use of ventricular assist devices (VADs). To date, there are no guidelines for appropriate selection for use of these devices that are approved by national societies in the field. This review addresses all of the general criteria for clinicians to keep in mind regarding when to refer a patient for evaluation and the specific issues addressed in patient selection. The field of mechanical circulatory support has advanced significantly over the past 10 years, resulting in rapid expansion of patients with advanced heart failure who can benefit from implantable devices. With progress of technology, limitations associated with age, body size, and comorbidities gradually become less prohibitive. The continuing simplification of design along with continued reduction in size of the devices, plus eventual elimination of the external drive line will make the use of VADs a superior option to heart transplant and even to medical management in many patients. We anticipate that the patient selection process outlined in the present review will continue to shift toward less advanced cases of heart failure. PMID:23290542

Miller, Leslie W; Guglin, Maya

2013-01-02

180

Selective autophagy.  

PubMed

During the last decade it has become evident that autophagy is not simply a non-selective bulk degradation pathway for intracellular components. On the contrary, the discovery and characterization of autophagy receptors which target specific cargo for lysosomal degradation by interaction with ATG8 (autophagy-related protein 8)/LC3 (light-chain 3) has accelerated our understanding of selective autophagy. A number of autophagy receptors have been identified which specifically mediate the selective autophagosomal degradation of a variety of cargoes including protein aggregates, signalling complexes, midbody rings, mitochondria and bacterial pathogens. In the present chapter, we discuss these autophagy receptors, their binding to ATG8/LC3 proteins and how they act in ubiquitin-mediated selective autophagy of intracellular bacteria (xenophagy) and protein aggregates (aggrephagy). PMID:24070473

Svenning, Steingrim; Johansen, Terje

2013-09-27

181

Lysosomal Phospholipase A2 and Phospholipidosis†  

PubMed Central

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

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

2006-01-01

182

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

PubMed

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

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

2012-07-20

183

In vitro endosome-lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver.  

PubMed

We have studied the endosome-lysosome transfer of internalized epidermal growth factor receptor (EGFR) complexes in a cell-free system from rat liver. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [(125)I]EGF was largely associated with a light endosomal fraction devoid of lysosomal markers. After an additional 30 min incubation in vitro in the presence of an ATP-regenerating system, the amount of [(125)I]EGF in this compartment decreased by 39%, with an increase in [(125)I]EGF in lysosomes. No transfer of [(125)I]EGF to the cytosol was detected. To assess the fate of the internalized EGFR protein over the time course of the endo-lysosomal transfer of the ligand, the effect of a saturating dose of native EGF on subsequent lysosomal targeting of the EGFR was evaluated by immunoblotting. A massive translocation of the EGFR to the endosomal compartment was observed in response to ligand injection coincident with its tyrosine phosphorylation and receptor recruitment of the tyrosine-phosphorylated adaptor protein Shc. During cell-free endosome-lysosome fusion, a time-dependent increase in the content of the EGFR and the two 55- and 46-kDa Shc isoforms was observed in lysosomal fractions with a time course superimposable with the lysosomal transfer of the ligand; no transfer of the 66-kDa Shc isoform was detected. The relationship between EGFR tyrosine kinase activity and EGFR sorting in endosomes investigated by immunoblot studies with anti-phosphotyrosine antibodies revealed that endosomal dephosphorylation of EGFR and Shc preceded lysosomal transfer. These results support the view that a lysosomal targeting machinery distinct from the endosomal receptor kinase activity, such as the recruitment of the signaling molecule Shc, may regulate this sorting event in the endosome. PMID:10561490

Authier, F; Chauvet, G

1999-11-12

184

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

PubMed Central

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

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

2012-01-01

185

Linking cell fate, trajectory choice, and target selection: genetic analysis of Sema-2b in olfactory axon targeting.  

PubMed

Neural circuit assembly requires selection of specific cell fates, axonal trajectories, and synaptic targets. By analyzing the function of a secreted semaphorin, Sema-2b, in Drosophila olfactory receptor neuron (ORN) development, we identified multiple molecular and cellular mechanisms that link these events. Notch signaling limits Sema-2b expression to ventromedial ORN classes, within which Sema-2b cell-autonomously sensitizes ORN axons to external semaphorins. Central-brain-derived Sema-2a and Sema-2b attract Sema-2b-expressing axons to the ventromedial trajectory. In addition, Sema-2b/PlexB-mediated axon-axon interactions consolidate this trajectory choice and promote ventromedial axon-bundle formation. Selecting the correct developmental trajectory is ultimately essential for proper target choice. These findings demonstrate that Sema-2b couples ORN axon guidance to postsynaptic target neuron dendrite patterning well before the final target selection phase, and exemplify how a single guidance molecule can drive consecutive stages of neural circuit assembly with the help of sophisticated spatial and temporal regulation. PMID:23719164

Joo, William J; Sweeney, Lora B; Liang, Liang; Luo, Liqun

2013-05-22

186

Multifingerprint based similarity searches for targeted class compound selection.  

PubMed

Molecular fingerprints are widely used for similarity-based virtual screening in drug discovery projects. In this paper we discuss the performance and the complementarity of nine two-dimensional fingerprints (Daylight, Unity, AlFi, Hologram, CATS, TRUST, Molprint 2D, ChemGPS, and ALOGP) in retrieving active molecules by similarity searching against a set of query compounds. For this purpose, we used biological data from HTS screening campaigns of four protein families (GPCRs, kinases, ion channels, and proteases). We have established threshold values for the similarity index (Tanimoto index) to be used as starting points for similarity searches. Based on the complementarities between the selections made by using different fingerprints we propose a multifingerprint approach as an efficient tool to balance the strengths and weaknesses of various fingerprints. PMID:16711740

Kogej, Thierry; Engkvist, Ola; Blomberg, Niklas; Muresan, Sorel

187

Increasing Intracellular Bioavailable Copper Selectively Targets Prostate Cancer Cells.  

PubMed

The therapeutic efficacy of two bis(thiosemicarbazonato) copper complexes, glyoxalbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(gtsm)] and diacetylbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(atsm)], for the treatment of prostate cancer was assessed in cell culture and animal models. Distinctively, copper dissociates intracellularly from Cu(II)(gtsm) but is retained by Cu(II)(atsm). We further demonstrated that intracellular H2gtsm [reduced Cu(II)(gtsm)] continues to redistribute copper into a bioavailable (exchangeable) pool. Both Cu(II)(gtsm) and Cu(II)(atsm) selectively kill transformed (hyperplastic and carcinoma) prostate cell lines but, importantly, do not affect the viability of primary prostate epithelial cells. Increasing extracellular copper concentrations enhanced the therapeutic capacity of both Cu(II)(gtsm) and Cu(II)(atsm), and their ligands (H2gtsm and H2atsm) were toxic only toward cancerous prostate cells when combined with copper. Treatment of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model with Cu(II)(gtsm) (2.5 mg/kg) significantly reduced prostate cancer burden (?70%) and severity (grade), while treatment with Cu(II)(atsm) (30 mg/kg) was ineffective at the given dose. However, Cu(II)(gtsm) caused mild kidney toxicity in the mice, associated primarily with interstitial nephritis and luminal distention. Mechanistically, we demonstrated that Cu(II)(gtsm) inhibits proteasomal chymotrypsin-like activity, a feature further established as being common to copper-ionophores that increase intracellular bioavailable copper. We have demonstrated that increasing intracellular bioavailable copper can selectively kill cancerous prostate cells in vitro and in vivo and have revealed the potential for bis(thiosemicarbazone) copper complexes to be developed as therapeutics for prostate cancer. PMID:23656859

Cater, Michael A; Pearson, Helen B; Wolyniec, Kamil; Klaver, Paul; Bilandzic, Maree; Paterson, Brett M; Bush, Ashley I; Humbert, Patrick O; La Fontaine, Sharon; Donnelly, Paul S; Haupt, Ygal

2013-05-24

188

Visual Cells Remember Earlier Applied Target: Plasticity of Orientation Selectivity  

PubMed Central

Background A canonical proposition states that, in mature brain, neurons responsive to sensory stimuli are tuned to specific properties installed shortly after birth. It is amply demonstrated that that neurons in adult visual cortex of cats are orientation-selective that is they respond with the highest firing rates to preferred oriented stimuli. Methodology/Principal Findings In anesthetized cats, prepared in a conventional fashion for single cell recordings, the present investigation shows that presenting a stimulus uninterruptedly at a non-preferred orientation for twelve minutes induces changes in orientation preference. Across all conditions orientation tuning curves were investigated using a trial by trial method. Contrary to what has been previously reported with shorter adaptation duration, twelve minutes of adaptation induces mostly attractive shifts, i.e. toward the adapter. After a recovery period allowing neurons to restore their original orientation tuning curves, we carried out a second adaptation which produced three major results: (1) more frequent attractive shifts, (2) an increase of their magnitude, and (3) an additional enhancement of responses at the new or acquired preferred orientation. Additionally, we also show that the direction of shifts depends on the duration of the adaptation: shorter adaptation in most cases produces repulsive shifts, whereas adaptation exceeding nine minutes results in attractive shifts, in the same unit. Consequently, shifts in preferred orientation depend on the duration of adaptation. Conclusion/Significance The supplementary response improvements indicate that neurons in area 17 keep a memory trace of the previous stimulus properties, thereby upgrading cellular performance. It also highlights the dynamic nature of basic neuronal properties in adult cortex since repeated adaptations modified both the orientation tuning selectivity and the response strength to the preferred orientation. These enhanced neuronal responses suggest that the range of neuronal plasticity available to the visual system is broader than anticipated.

Ghisovan, Narcis; Nemri, Abdellatif; Shumikhina, Svetlana; Molotchnikoff, Stephane

2008-01-01

189

Classification of Subcellular Location by Comparative Proteomic Analysis of Native and Density-shifted Lysosomes*  

PubMed Central

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.

Della Valle, Maria Cecilia; Sleat, David E.; Zheng, Haiyan; Moore, Dirk F.; Jadot, Michel; Lobel, Peter

2011-01-01

190

Bibliomics-based Selection of Analgesics Targets through Google-PageRank-like Algorithm  

Microsoft Academic Search

To screen for effective and non-addictive pain-killers in substitution of morphine, appropriate drug targets should be selected firstly. They can be retrieved through transcriptomic, proteomic, bibliomic and any other high-throughput technique. In the view of bibliomics, the known or potential analgesics targets are hidden in all human genes (proteins) cited in MED-LINE entries with \\

Lun Yang; Bin Wang; Gongli Xia; Zhenhua Xia; Langlai Xu

2007-01-01

191

Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins  

Microsoft Academic Search

Cytosine-5 DNA methylation is a critical signal defining heritable epigenetic states of transcription. As aberrant methylation patterns often accompany disease states, the ability to target cytosine methyl- ation to preselected regions could prove valuable in re-establishing proper gene regulation. We employ the strategy of targeted gene methylation in yeast, which has a naturally unmethylated genome, select- ively directing de novo

Christopher D. Carvin; Rebecca D. Parr; Michael P. Kladde

2003-01-01

192

In vivo biotinylated proteins as targets for phage-display selection experiments  

Microsoft Academic Search

Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins. Two critical components of a successful selection experiment are having a pure target protein and its immobilization in a native conformation. To achieve both of these requirements in a single step, we have devised cytoplasmic expression vectors for expression of proteins that are tagged at

Michael D Scholle; Frank R Collart; Brian K Kay

2004-01-01

193

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

ERIC Educational Resources Information Center

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

Smith, Nicholas A.; Trainor, Laurel J.

2011-01-01

194

A generic video and radar data fusion system for improved target selection  

Microsoft Academic Search

This paper presents an automotive video and radar data fusion framework that can be used as a preliminary stage of an automatic cruise control or collision mitigation by braking system. The fusion framework finds the optimal assignment of radar and camera target reports and provides improved state estimates for the fused targets. A sophisticated critical path selection is presented and

Dennis Muller; Josef Pauli; Mirko Meuter; Lali Ghosh; Stefan Muller-Schneiders

2011-01-01

195

Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion  

Microsoft Academic Search

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells

Benjamin Misselwitz; Naomi Barrett; Saskia Kreibich; Pascale Vonaesch; Daniel Andritschke; Samuel Rout; Kerstin Weidner; Milos Sormaz; Pascal Songhet; Peter Horvath; Mamta Chabria; Viola Vogel; Doris M. Spori; Patrick Jenny; Wolf-Dietrich Hardt

2012-01-01

196

Experimentally induced lysosomal dysfunction disrupts processing of hypothalamic releasing factors.  

PubMed

Previous studies have shown that experimentally induced lysosomal dysfunction elicits various features of aging in the cortical telencephalon. The present study used cultured slices to test if: (1) it causes similar changes in the hypothalamus, and/or (2) modifies the processing of two releasing factors important to aging. A 2-day exposure to N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), a selective inhibitor of cathepsins B and L, triggered a pronounced increase in the numbers of lysosomes in the ventromedial and dorsomedial nuclei, and in lateral hypothalamus. Continued incubation with the inhibitor for 3-12 days resulted in the spread of endosomes-lysosomes into dendrites and, in the lateral hypothalamus, the formation of massive, lysosome-filled expansions of neuronal processes (meganeurites). These effects did not occur in the arcuate nucleus, making it the first region so far examined in which lysosomal proliferation is not initiated by hydrolase inhibitors. Despite this, a dense plexus of axons and terminals in the median eminence was partially depleted of growth hormone releasing hormone (GHRH) within 48 hours after addition of ZPAD. Moreover, the inhibitor caused axonal GHRH to become collected into large puncta, an effect highly suggestive of a partial failure in axonal transport. GHRH mRNA levels were not greatly affected by 6 days of ZPAD exposure, indicating that reduced expression did not play a major role in the peptide changes seen at 48 hours. Similar but less pronounced immunocytochemical changes were recorded for the somatostatin system in the arcuate and periventricular nucleus. It is concluded that lysosome dysfunction: (1) has different consequences for the arcuate nucleus than other brain regions, and (2) disrupts transport of hypothalamic releasing factors. The potential significance of the results to endocrine senescence is discussed. PMID:9811115

Bi, X; Pinkstaff, J; Nguyen, K; Gall, C M; Lynch, G

1998-11-23

197

Specific lysosomal transport of small neutral amino acids  

SciTech Connect

Studies of amino acid exodus from lysosomes have allowed us previously to describe transport systems specific for cystine and another for cationic amino acids in fibroblast lysosomes. They are now able to study amino acid uptake into highly purified fibroblast lysosomes obtained by separating crude granular fraction on gradients formed by centrifugation in 35% isoosmotic Percoll solutions. Analog inhibition and saturation studies indicate that L-(/sup 14/C)proline (50 ..mu..M) uptake by fibroblast lysosomes at 37/sup 0/C in 50 mM citrate/tris pH 7.0 buffer containing 0.25 M sucrose is mediated by two transport systems, one largely specific for L-proline and the other for which transport is shared with small neutral amino acids such as alanine, serine and threonine. At 7 mM, L-proline inhibits L-(/sup 14/C)proline uptake almost completely, whereas ala, ser, val, thr, gly, N-methylalanine and sarcosine inhibit proline uptake by 50-65%. The system shared by alanine, serine and threonine is further characterized by these amino acids strongly inhibiting the uptakes of each other. Lysosomal proline transport is selective for the L-isomer of the amino acid, and is scarcely inhibited by 7 mM arg, glu, asp, leu, phe, his, met, (methylamino) isobutyrate, betaine or N,N-dimethylglycine. Cis or trans-4-hydroxy-L-proline inhibit proline uptake only slightly. In sharp contrast to the fibroblast plasma membrane in which Na/sup +/ is required for most proline and alanine transport, lysosomal uptake of these amino acids occurs independently of Na/sup +/.

Pisoni, R.L.; Flickinger, K.S.; Thoene, J.G.; Christensen, H.N.

1986-05-01

198

Newborn screening for neuropathic lysosomal storage disorders.  

PubMed

Interest in newborn screening (NBS) for lysosomal storage disorders (LSDs) has increased significantly due to newly developed enzyme replacement therapy (ERT), the need for early diagnosis, and advances in technical developments. Since the central nervous system cannot be treated by ERT, neuronopathic LSDs are generally not the primary target of NBS. An exception is Krabbe disease, in which hematopoietic stem cell transplantation before the onset of symptoms has benefits. However, NBS for LSD relies on measuring enzyme activities, so the most severely affected individuals (usually patients with neuronopathic subtypes) will be detected together with patients with less severe disease. In the near future, NBS is likely to be developed for diseases such as Gaucher, Niemann-Pick A/B, and certain mucopolysaccharidoses. The ability to predict phenotypes (neuronopathic or not) by enzyme activity and genotyping will therefore be critical for adequate patient management. This article reviews the status of LSD screening and issues concerning detection of neuronopathic LSDs by screening. PMID:20532820

Hwu, Wuh-Liang; Chien, Yin-Hsiu; Lee, Ni-Chung

2010-06-08

199

Human lysosomal ?-mannosidases exhibit different inhibition and metal binding properties  

PubMed Central

Two structurally-related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal ?-mannosidase (hLM, MAN2B1) and the human core ?-1, 6-specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II ?-mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine-based compounds designed for selective inhibition of Golgi ?-mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal ?-mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.

Venkatesan, Meenakshi; Kuntz, Douglas A; Rose, David R

2009-01-01

200

Differential actions of insecticides on target sites: basis for selective toxicity  

Microsoft Academic Search

Whereas the selective toxicity of insecticides between insects and mammals has a long history of studies, it is now becoming abundantly clear that, in many cases, the differential action of insecticides on insects and mammalian target receptor sites is an important factor. In this paper, we first introduce the mechanism of action and the selective toxicity of pyrethroids as a

T. Narahashi; X. Zhao; T. Ikeda; K. Nagata; JZ Yeh

2007-01-01

201

Selective cavitand-mediated endocytosis of targeted imaging agents into live cells.  

PubMed

A water-soluble synthetic receptor molecule is capable of selective, controlled endocytosis of a specifically tagged target molecule in different types of living human cells. The presence of suitable choline-derived binding handles is essential for the molecular recognition and transport process, allowing selective guest transport and imaging of cancer cells. PMID:23621383

Ghang, Yoo-Jin; Schramm, Michael P; Zhang, Fan; Acey, Roger A; David, Clement N; Wilson, Emma H; Wang, Yinsheng; Cheng, Quan; Hooley, Richard J

2013-04-30

202

Autophagosome maturation and lysosomal fusion.  

PubMed

Compartmentalization is essential in the eukaryotic cell and this is most often achieved by sequestering specific components that perform a related function in a membrane-bound organelle. To function normally these organelles must transiently fuse with other compartments in order to transfer protein and lipid that is needed for them to function. These events must be highly coordinated otherwise non-specific fusion could occur leading to loss of compartment identity and function. The autophagosome is a specialized membrane compartment that delivers cytosolic components to the lysosome for degradation. Likewise, this delivery is coordinated so that only when the autophagosome is fully formed is it imparted with the information to allow it to specifically fuse with the endocytic system and deliver its contents to the lysosome. In the present chapter, I discuss our current understanding of how this happens. PMID:24070472

Ganley, Ian G

2013-09-27

203

Selection of Cyclic-Peptide Inhibitors Targeting Aurora Kinase A: Problems and Solutions  

PubMed Central

The critical role of Aurora kinase in cell cycle progression and its deregulation in cancer has garnered significant interest. As such, numerous Aurora targeted inhibitors have been developed to date, almost all of which target the ATP cleft at the active site. These current inhibitors display polypharmacology; that is, they target multiple kinases, and some are being actively pursued as therapeutics. Currently, there are no general approaches for targeting Aurora at sites remote from the active site, which in the long term may provide new insights regarding the inhibition of Aurora as well as other protein kinases, and provide pharmacological tools for dissecting Aurora kinase biology. Toward this long term goal, we have recently developed a bivalent selection strategy that allows for the identification of cyclic peptides that target the surface of PKA, while the active site is blocked by an ATP-competitive compound. Herein, we extend this approach to Aurora kinase (Aurora A), which required significant optimization of selection conditions to eliminate background peptides that target the streptavidin matrix upon which the kinases are immobilized. Using our optimized selection conditions, we have successfully selected several cyclic peptide ligands against Aurora A. Two of these inhibitors demonstrated IC50 values of 10 ?M and were further interrogated. The CTRPWWLC peptide was shown to display a noncompetitive mode of inhibition suggesting that alternate sites on Aurora beyond the ATP and peptide substrate binding site may be potentially targeted.

Shomin, Carolyn D.; Restituyo, Elizabeth; Cox, Kurt J.; Ghosh, Indraneel

2011-01-01

204

Lysosomal acid lipase-deficient mice: depletion of white and brown fat, severe hepatosplenomegaly, and shortened life span  

Microsoft Academic Search

Lysosomal acid lipase (LAL) is essential for the hydrolysis of triglycerides (TG) and cholesteryl esters (CE) in lysosomes. A mouse model created by gene targeting pro- duces no LAL mRNA, protein, or enzyme activity. The lal 2 \\/ 2 mice appear normal at birth, survive into adult- hood, and are fertile. Massive storage of TG and CE is ob- served

Hong Du; Martin Heur; Ming Duanmu; Gregory A. Grabowski; David Y. Hui; David P. Witte; Jaya Mishra

205

Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid-binding peptide.  

PubMed

Atwo-step targeting strategy was used to identify improved laccases for bleaching carotenoid-containing stains on fabric. We first applied a modified phage display technique to identify peptide sequences capable of binding specifically to carotenoid stains and not to fabric. Prior deselection on the support on which the carotenoid was localized, increased stringency during the biopanning target selection process, and analysis of the phage peptides' binding to the target after acid elution and polymerase chain reaction (PCR) postacid elution, were used to isolate phage peptide libraries with increased binding selectivity and affinity. Peptide sequences were selected based on identified consensus motifs. We verified the enhanced carotenoid-binding properties of the peptide YGYLPSR and subsequently cloned and expressed C-terminal variants of laccase from Stachybotrys chartarum containing carotenoid-binding peptides YGYLPSR, IERSAPATAPPP, KASAPAL, CKASAPALC, and SLLNATK. These targeted peptide-laccase fusions demonstrate enhanced catalytic properties on stained fabrics. PMID:15200474

Janssen, G G; Baldwin, T M; Winetzky, D S; Tierney, L M; Wang, H; Murray, C J

2004-07-01

206

Glycoproteins of the lysosomal membrane  

Microsoft Academic Search

Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono- and\\/ or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse mono- clonal antibody to the 120-kD antigen

VICTORIA LEWIS; SAMUEL A. GREEN; MARK MARSH; ARI HELENIUS; IRA MELLMAN

1985-01-01

207

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

PubMed Central

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

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

2012-01-01

208

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

PubMed Central

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

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

2010-01-01

209

FOXP2 targets show evidence of positive selection in European populations.  

PubMed

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

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

2013-04-18

210

Signaling from lysosomes to mitochondria sensitizes cancer cells to photodynamic treatment  

Microsoft Academic Search

Previously, we showed that photosensitizers that localize to lysosomes are more effective in killing cancer cells than ones directed to mitochondria after photodynamic treatment (PDT). The photosensitizer, phthalocyanine 4 (Pc 4), localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. However, analogues of Pc 4 (e.g., Pc 181) that primarily target lysosomes still produce mitochondria-mediated cell

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

2011-01-01

211

Lytic granules, secretory lysosomes and disease.  

PubMed

Lytic granules harbour many of the dangerous apoptosis-inducing molecules of the immune system, including perforin, granzymes and Fas ligand. Safe transport, storage and release of these lytic components is vital. As a secretory lysosome, the lytic granule is able to accomplish these roles, as well as conferring the lysosomal functions of cytotoxic T lymphocytes and natural killer cells. Secretory lysosomes are common to many other haemopoietic cells and also melanocytes. Many of the proteins used in lysosomal secretion are found in both melanocytes and hemopoietic cells, and are dysfunctional in genetic diseases with defects in these proteins. The genetically heterogeneous Hermansky-Pudlak syndrome represents an excellent model for revealing proteins involved in secretory lysosome functioning. However, studies of this disease reveal differences between the various different types of secretory lysosomes, including lytic granules. PMID:14499259

Clark, Richard; Griffiths, Gillian M

2003-10-01

212

Identification of two lysosomal membrane glycoproteins  

Microsoft Academic Search

TWO murine lysosome-assoeiated membrane proteins, LAMP-1 of 105,000- 115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chao- tropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to

JEFF W. CHEN; THERESA L. MURPHY; MARK C. WlLLINGHAM; IRA PASTAN; J. THOMAS AUGUST

1985-01-01

213

Immune system irregularities in lysosomal storage disorders  

Microsoft Academic Search

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

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

2008-01-01

214

Different Steady State Subcellular Distributions of the Three Splice Variants of Lysosome-associated Membrane Protein LAMP2 Are Determined Largely by the COOH-terminal Amino Acid Residue  

Microsoft Academic Search

The extensively glycosylated lysosome-asso- ciated membrane proteins (LAMP)-2a, b, and c are de- rived from a single gene by alternative splicing that pro- duces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at

Nancy R. Gough; Douglas M. Fambrough

1997-01-01

215

Chaperone-mediated autophagy: a unique way to enter the lysosome world  

PubMed Central

All cellular proteins undergo continuous synthesis and degradation. This permanent renewal is necessary to maintain a functional proteome and to allow for rapid changes in levels of specific proteins with regulatory purposes. Although for a long time lysosomes were considered unable to contribute to the selective degradation of individual proteins, the discovery of chaperone-mediated autophagy (CMA) changed this notion. Here, we review the characteristics that set CMA apart from other types of lysosomal degradation and the subset of molecules that confer cells the capability to identify individual cytosolic proteins and direct them across the lysosomal membrane for degradation.

Kaushik, Susmita; Cuervo, Ana Maria

2012-01-01

216

Selection of IFE target materials from a safety and environmental perspective  

NASA Astrophysics Data System (ADS)

Target materials for inertial fusion energy (IFE) power plant designs might be selected for a wide variety of reasons including wall absorption of driver energy, material opacity, cost and ease of fabrication. While each of these issues are of great importance, target materials should also be selected based upon their safety and environmental (S&E) characteristics. The present work focuses on the recycling, waste management and accident dose characteristics of potential target materials. If target materials are recycled so that the quantity is small, isotopic separation may be economically viable. Therefore, calculations have been completed for all stable isotopes for all elements from lithium to polonium. The results of these calculations are used to identify specific isotopes and elements that are most likely to be offensive as well as those most likely to be acceptable in terms of their S&E characteristics.

Latkowski, J. F.; Sanz, J.; Reyes, S.; Gomez del Rio, J.

2001-05-01

217

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

PubMed Central

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

Goker, Markus; Klenk, Hans-Peter

2013-01-01

218

Targeted gene disruption in Cryptococcus neoformans using double-joint PCR with split dominant selectable markers.  

PubMed

Cryptococcus neoformans causes fatal meningoencephalitis if not timely treated. Targeted gene disruption for functional analysis of a gene involves overlap PCR for the production of gene disruption cassettes carrying dominant selectable markers, followed by biolistic transformation. However, the conventional overlap PCR method between two flanking regions of the target gene and selectable marker is often inefficient due to the long length of the PCR product and the presence of multiple templates. Here we describe double-joint PCR with split dominant selectable markers for the more convenient generation of a gene-disruption cassette in C. neoformans with high targeted integration frequency (Kim et al., Biochem. Biophys. Res. Commun 390(3):983-988, 2009). PMID:22328368

Kim, Min Su; Kim, Seo-Young; Jung, Kwang-Woo; Bahn, Yong-Sun

2012-01-01

219

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

PubMed

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

Göker, Markus; Klenk, Hans-Peter

2013-05-20

220

Target selection and accessibility for rendezvous with a Near-Earth asteroid mission  

Microsoft Academic Search

Mission to asteroids and comets has been the hot spot of deep space exploration in the new century. The choice of a suitable\\u000a target, which involves both scientific value and technical feasibility, becomes a difficult task to accomplish due to limited\\u000a energy and technology. The aim of this paper is to provide an approach to selecting a target and evaluating

Dong Qiao; Pingyuan Cui; Hutao Cui

2007-01-01

221

Dissociable dopaminergic control of saccadic target selection and its implications for reward modulation.  

PubMed

To investigate mechanisms by which reward modulates target selection, we studied the behavioral effects of perturbing dopaminergic activity within the frontal eye field (FEF) of monkeys performing a saccadic choice task and simulated the effects using a plausible cortical network. We found that manipulation of FEF activity either by blocking D1 receptors (D1Rs) or by stimulating D2 receptors (D2Rs) increased the tendency to choose targets in the response field of the affected site. However, the D1R manipulation decreased the tendency to repeat choices on subsequent trials, whereas the D2R manipulation increased that tendency. Moreover, the amount of shift in target selection resulting from the two manipulations correlated in opposite ways with the baseline stochasticity of choice behavior. Our network simulation results suggest that D1Rs influence target selection mainly through their effects on the strength of inputs to the FEF and on recurrent connectivity, whereas D2Rs influence the excitability of FEF output neurons. Altogether, these results reveal dissociable dopaminergic mechanisms influencing target selection and suggest how reward can influence adaptive choice behavior via prefrontal dopamine. PMID:23401524

Soltani, Alireza; Noudoost, Behrad; Moore, Tirin

2013-02-11

222

Dissociable dopaminergic control of saccadic target selection and its implications for reward modulation  

PubMed Central

To investigate mechanisms by which reward modulates target selection, we studied the behavioral effects of perturbing dopaminergic activity within the frontal eye field (FEF) of monkeys performing a saccadic choice task and simulated the effects using a plausible cortical network. We found that manipulation of FEF activity either by blocking D1 receptors (D1Rs) or by stimulating D2 receptors (D2Rs) increased the tendency to choose targets in the response field of the affected site. However, the D1R manipulation decreased the tendency to repeat choices on subsequent trials, whereas the D2R manipulation increased that tendency. Moreover, the amount of shift in target selection resulting from the two manipulations correlated in opposite ways with the baseline stochasticity of choice behavior. Our network simulation results suggest that D1Rs influence target selection mainly through their effects on the strength of inputs to the FEF and on recurrent connectivity, whereas D2Rs influence the excitability of FEF output neurons. Altogether, these results reveal dissociable dopaminergic mechanisms influencing target selection and suggest how reward can influence adaptive choice behavior via prefrontal dopamine.

Soltani, Alireza; Noudoost, Behrad; Moore, Tirin

2013-01-01

223

Selective and efficient retardation of cancers expressing cytoskeleton-associated protein 2 by targeted RNA replacement.  

PubMed

Human cytoskeleton-associated protein 2 (hCKAP2) is upregulated and highly expressed in various human malignances. hCKAP2 has microtubule-stabilizing characteristics and potentially regulates the dynamics and assembly of the mitotic spindle and chromosome segregation, indicating that hCKAP2 plays important functions during mitosis. In this study, we evaluated hCKAP2 as a plausible anticancer target through development and validation of a targeted cancer gene therapy strategy based on targeting and replacement of hCKAP2 RNA using a trans-splicing ribozyme. This targeted RNA replacement triggered transgene activity via accurate trans-splicing reaction selectively in human cancer cells expressing the hCKAP2 RNA and simultaneously reduced the expression level of the RNA in the cells. Adenoviral vector encoding the hCKAP2-specific trans-splicing ribozyme selectively induced cytotoxicity in tumor cells expressing hCKAP2. Moreover, intratumoral injection of the virus produced selective and efficient regression of tumor that had been subcutaneously inoculated with hCKAP2-positive colon cancer cells in mice with minimal liver toxicity. Furthermore, orthotopically multifocal hCKAP2-positive hepatocarcinoma established in mice were efficiently regressed by systemic delivery of adenoviral vector encoding the specific ribozyme under the control of a liver-selective phosphoenolpyruvate carboxykinase promoter with least hepatotoxicity. The results indicate that hCKAP2 RNA is a promising target for anticancer approach based on trans-splicing ribozyme-mediated RNA replacement. PMID:21328343

Ban, Guyee; Jeong, Jin-Sook; Kim, Areum; Kim, Sung Jin; Han, Sang-Young; Kim, In-Hoo; Lee, Seong-Wook

2011-04-13

224

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

PubMed Central

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

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

2009-01-01

225

Ultrastructural Analysis of Neuronal and Non-neuronal Lysosomal Storage in Mucolipidosis Type II Knock-in Mice.  

PubMed

Abstract The GlcNAc-1-phosphotransferase catalyzes the first step in the formation of mannose 6-phosphate (M6P) residues on lysosomal acid hydrolases that is essential for the efficient transport of newly synthesized lysosomal enzymes to lysosomes and the maintenance of lysosomal functions. Mutations in the GlcNAc-1-phosphotransferase cause the lysosomal storage disease mucolipidosis type II (MLII), resulting in mistargeting and hypersecretion of multiple lysosomal hydrolases and subsequent lysosomal accumulation of nondegraded material in several tissues. To describe cell-type specificity, compositional differences, and subcellular distribution of the stored material we performed an in-depth ultrastructural analysis of lysosomal storage in brain and retina of MLII knock-in mice using electron microscopy. Massive vacuoles filled with heterogeneous storage material have been found in the soma, swollen axons, and dendrites of Purkinje, and granular cells in 9-month-old MLII mice. In addition, non-neuronal cells, such as microglial, astroglial, and endothelial cells, exhibit storage material. Fucose-specific lectin histochemistry demonstrated the accumulation of fucose-containing oligosaccharides, indicating that targeting of the lysosomal ?-fucosidase is strongly impaired in all cerebellar cell types. The data suggest that the accumulation of storage material might affect neuronal function and survival in a direct cell-autonomous manner, as well as indirectly by disturbed metabolic homeostasis between glial and neuronal cells or by cerebrovascular complications. PMID:24047352

Schweizer, Michaela; Markmann, Sandra; Braulke, Thomas; Kollmann, Katrin

2013-10-01

226

Artesunate Activates Mitochondrial Apoptosis in Breast Cancer Cells via Iron-catalyzed Lysosomal Reactive Oxygen Species Production*  

PubMed Central

The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment.

Hamacher-Brady, Anne; Stein, Henning A.; Turschner, Simon; Toegel, Ina; Mora, Rodrigo; Jennewein, Nina; Efferth, Thomas; Eils, Roland; Brady, Nathan R.

2011-01-01

227

Sunitinib and SU11652 Inhibit Acid Sphingomyelinase, Destabilize Lysosomes, and Inhibit Multidrug Resistance.  

PubMed

Defective apoptosis signaling and multidrug resistance are major barriers for successful cancer treatment. To identify drugs capable of targeting treatment-resistant cancer cells, we screened small-molecule kinase inhibitor libraries for compounds that decrease the viability of apoptosis-resistant human MCF7-Bcl-2 breast cancer cells. SU11652, a multitargeting receptor tyrosine kinase inhibitor, emerged as the most potent compound in the screen. In addition to MCF7-Bcl-2 cells, it effectively killed HeLa cervix carcinoma, U-2-OS osteosarcoma, Du145 prostate carcinoma, and WEHI-S fibrosarcoma cells at low micromolar concentration. SU11652 accumulated rapidly in lysosomes and disturbed their pH regulation and ultrastructure, eventually leading to the leakage of lysosomal proteases into the cytosol. Lysosomal destabilization was preceded by an early inhibition of acid sphingomyelinase, a lysosomal lipase that promotes lysosomal membrane stability. Accordingly, Hsp70, which supports cancer cell survival by increasing lysosomal acid sphingomyelinase activity, conferred partial protection against SU11652-induced cytotoxicity. Remarkably, SU11652 killed multidrug-resistant Du145 prostate cancer cells as effectively as the drug-sensitive parental cells, and subtoxic concentrations of SU11652 effectively inhibited multidrug-resistant phenotype in Du145 prostate cancer cells. Notably, sunitinib, a structurally almost identical and widely used antiangiogenic cancer drug, exhibited similar lysosome-dependent cytotoxic activity, albeit with significantly lower efficacy. The significantly stronger lysosome-targeting activity of SU11652 suggests that it may display better efficacy in cancer treatment than sunitinib, encouraging further evaluation of its anticancer activity in vivo. Furthermore, our data provide a rationale for novel approaches to target drug-resistant cancers by combining classic chemotherapy with sunitinib or SU11652. Mol Cancer Ther; 12(10); 2018-30. ©2013 AACR. PMID:23920274

Ellegaard, Anne-Marie; Groth-Pedersen, Line; Oorschot, Viola; Klumperman, Judith; Kirkegaard, Thomas; Nylandsted, Jesper; Jäättelä, Marja

2013-08-06

228

Attention blinks for selection, not perception or memory: reading sentences and reporting targets.  

PubMed

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 attentional blink, and does it affect both tasks? Here, sentence report was combined with report of two target words (Experiments 1 and 2) or two inserted target digits, Arabic numerals or word digits (Experiments 3 and 4). When participants reported only the targets an attentional blink was always observed. When they reported both the sentence and targets, sentence report was quite accurate but there was an attentional blink in picking out the targets when they were part of the sentence. When targets were extra digits inserted in the sentence there was no blink when viewers also reported the sentence. These results challenge some theories of the attentional blink: Blinks result from online selection, not perception or memory. PMID:22022894

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

2011-10-24

229

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

PubMed Central

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

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

2007-01-01

230

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

PubMed

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

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

2007-04-16

231

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

NASA Astrophysics Data System (ADS)

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.

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

232

Frontoparietal theta activity supports behavioral decisions in movement-target selection  

PubMed Central

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

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

2012-01-01

233

Lysosomotropic Properties of Weakly Basic Anticancer Agents Promote Cancer Cell Selectivity In Vitro  

PubMed Central

Drug distribution in cells is a fundamentally important, yet often overlooked, variable in drug efficacy. Many weakly basic anticancer agents accumulate extensively in the acidic lysosomes of normal cells through ion trapping. Lysosomal trapping reduces the activity of anticancer drugs, since anticancer drug targets are often localized in the cell cytosol or nucleus. Some cancer cells have defective acidification of lysosomes, which causes a redistribution of trapped drugs from the lysosomes to the cytosol. We have previously established that such differences in drug localization between normal and cancer cells can contribute to the apparent selectivity of weakly basic drugs to cancer cells in vitro. In this work, we tested whether this intracellular distribution-based drug selectivity could be optimized based on the acid dissociation constant (pKa) of the drug, which is one of the determinants of lysosomal sequestration capacity. We synthesized seven weakly basic structural analogs of the Hsp90 inhibitor geldanamycin (GDA) with pKa values ranging from 5 to 12. The selectivity of each analog was expressed by taking ratios of anti-proliferative IC50 values of the inhibitors in normal fibroblasts to the IC50 values in human leukemic HL-60 cells. Similar selectivity assessments were performed in a pair of cancer cell lines that differed in lysosomal pH as a result of siRNA-mediated alteration of vacuolar proton ATPase subunit expression. Optimal selectivity was observed for analogs with pKa values near 8. Similar trends were observed with commercial anticancer agents with varying weakly basic pKa values. These evaluations advance our understanding of how weakly basic properties can be optimized to achieve maximum anticancer drug selectivity towards cancer cells with defective lysosomal acidification in vitro. Additional in vivo studies are needed to examine the utility of this approach for enhancing selectivity.

Ndolo, Rosemary A.; Luan, Yepeng; Duan, Shaofeng; Forrest, M. Laird; Krise, Jeffrey P.

2012-01-01

234

Lysosomotropic properties of weakly basic anticancer agents promote cancer cell selectivity in vitro.  

PubMed

Drug distribution in cells is a fundamentally important, yet often overlooked, variable in drug efficacy. Many weakly basic anticancer agents accumulate extensively in the acidic lysosomes of normal cells through ion trapping. Lysosomal trapping reduces the activity of anticancer drugs, since anticancer drug targets are often localized in the cell cytosol or nucleus. Some cancer cells have defective acidification of lysosomes, which causes a redistribution of trapped drugs from the lysosomes to the cytosol. We have previously established that such differences in drug localization between normal and cancer cells can contribute to the apparent selectivity of weakly basic drugs to cancer cells in vitro. In this work, we tested whether this intracellular distribution-based drug selectivity could be optimized based on the acid dissociation constant (pKa) of the drug, which is one of the determinants of lysosomal sequestration capacity. We synthesized seven weakly basic structural analogs of the Hsp90 inhibitor geldanamycin (GDA) with pKa values ranging from 5 to 12. The selectivity of each analog was expressed by taking ratios of anti-proliferative IC(50) values of the inhibitors in normal fibroblasts to the IC(50) values in human leukemic HL-60 cells. Similar selectivity assessments were performed in a pair of cancer cell lines that differed in lysosomal pH as a result of siRNA-mediated alteration of vacuolar proton ATPase subunit expression. Optimal selectivity was observed for analogs with pKa values near 8. Similar trends were observed with commercial anticancer agents with varying weakly basic pKa values. These evaluations advance our understanding of how weakly basic properties can be optimized to achieve maximum anticancer drug selectivity towards cancer cells with defective lysosomal acidification in vitro. Additional in vivo studies are needed to examine the utility of this approach for enhancing selectivity. PMID:23145164

Ndolo, Rosemary A; Luan, Yepeng; Duan, Shaofeng; Forrest, M Laird; Krise, Jeffrey P

2012-11-07

235

Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl- symport.  

PubMed Central

We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53-59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomyces and Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30-120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.

Ohkuma, S; Sato, T; Okamoto, M; Matsuya, H; Arai, K; Kataoka, T; Nagai, K; Wasserman, H H

1998-01-01

236

Near surface swimming of Salmonella Typhimurium explains target-site selection and cooperative invasion.  

PubMed

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ?1.5-3 s in "near surface swimming". This increased the local pathogen density and facilitated "scanning" of the host surface topology. We observed transient TTSS-1 and fim-independent "stopping" and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria. PMID:22911370

Misselwitz, Benjamin; Barrett, Naomi; Kreibich, Saskia; Vonaesch, Pascale; Andritschke, Daniel; Rout, Samuel; Weidner, Kerstin; Sormaz, Milos; Songhet, Pascal; Horvath, Peter; Chabria, Mamta; Vogel, Viola; Spori, Doris M; Jenny, Patrick; Hardt, Wolf-Dietrich

2012-07-26

237

Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion  

PubMed Central

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ?1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria.

Kreibich, Saskia; Vonaesch, Pascale; Andritschke, Daniel; Rout, Samuel; Weidner, Kerstin; Sormaz, Milos; Songhet, Pascal; Horvath, Peter; Chabria, Mamta; Vogel, Viola; Spori, Doris M.; Jenny, Patrick; Hardt, Wolf-Dietrich

2012-01-01

238

Autophagy Regulates Cholesterol Efflux from Macrophage Foam Cells via Lysosomal Acid Lipase  

PubMed Central

SUMMARY The lipid droplet (LD) is the major site of cholesterol storage in macrophage foam cells and is a potential therapeutic target for the treatment of atherosclerosis. Cholesterol, stored as cholesteryl esters (CEs), is liberated from this organelle and delivered to cholesterol acceptors. The current paradigm attributes all cytoplasmic CE hydrolysis to the action of neutral CE hydrolases. Here, we demonstrate an important role for lysosomes in LD CE hydrolysis in cholesterol-loaded macrophages, in addition to that mediated by neutral hydrolases. Furthermore, we demonstrate that LDs are delivered to lysosomes via autophagy, where lysosomal acid lipase (LAL) acts to hydrolyze LD CE to generate free cholesterol mainly for ABCA1-dependent efflux; this process is specifically induced upon macrophage cholesterol loading. We conclude that, in macrophage foam cells, lysosomal hydrolysis contributes to the mobilization of LD-associated cholesterol for reverse cholesterol transport.

Ouimet, Mireille; Franklin, Vivian; Mak, Esther; Liao, Xianghai; Tabas, Ira; Marcel, Yves L.

2012-01-01

239

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

Microsoft Academic Search

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

Shavin Malhotra; K. Sivakumar; PengCheng Zhu

2009-01-01

240

Laser polarization and reflectance characterization of selected target and background material  

Microsoft Academic Search

This paper describes the relative polarization and reflectance characterization of background and selected target items to demonstrate the differences material type and source wavelength have on these measurements. The advanced reflectance and polarization instrument (ARPI) was modified to allow three lasers with different wavelengths to be used. This allowed for similar spot size, location, and angles to be used to

Hollis H. Bennett Jr.; Morris P. Fields; Zenon Derzko

2006-01-01

241

Studies on the idea selection and target mode of marketing in public utility organizations  

Microsoft Academic Search

At the present time, the researches of marketing in public utility organizations lagged behind that in the field of enterprise significantly. It's still lack of rational understanding and systemic illustration on those problems, such as whether we need to marketing, how to select the marketing idea and marketing target, and so on. The authors put forward that marketing in public

Zhu Jing; Wang Hong-yu

2011-01-01

242

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

NASA Astrophysics Data System (ADS)

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 ~105 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.

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

2013-10-01

243

Lysosomal cysteine proteases: more than scavengers  

Microsoft Academic Search

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

Boris Turk; Dušan Turk; Vito Turk

2000-01-01

244

Animal models of lysosomal disease: An overview  

Microsoft Academic Search

The relative rarity of human lysosomal disorders, extremely heterogeneous genetic background and ethical restrictions make well-controlled studies difficult with human patients. Genetically authentic animal models complement human patients with their ready availability, homogeneous genetic background and the relatively flexible experimental designs. Spontaneous animal models of human lysosomal disorders are rare, particularly among small laboratory animals. However, the homologous recombination and

K. Suzuki; J.-E. Månsson

1998-01-01

245

The lysosome or lysosome-related organelle may serve as a Ca2+ store in the boutons of hippocampal pyramidal cells.  

PubMed

Boutons are specialised presynaptic compartments that lie along the axons of central neurons. Release of neurotransmitter from boutons is tightly regulated by the level of intracellular calcium [Ca2+]i. A rise in Ca2+ level may be generated in several ways; entry of extracellular Ca2+ via voltage gated calcium channels (VGCCs), entry via ligand-operated channels (LOCs) or the release of Ca2+ from intracellular Ca2+ stores. The role of Ca2+ stores in boutons remains poorly understood, despite recent work indicating that the release of Ca2+ from the endoplasmic reticulum (ER) may contribute to transmitter release. In this study we assess whether the lysosome or a closely related organelle functions as a Ca2+ store in the boutons of hippocampal pyramidal neurones. Lysosomes are small acidic organelles more commonly known for their role in degrading redundant cellular constituents. Using a fluorescent lysosomal marker, we show that lysosomes are located in the axons of hippocampal CA3 neurones. Selective pharmacological lysis of the lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) generates rapid, highly focal Ca2+ transients within the axon and increases the frequency of spontaneous miniature excitatory post-synaptic currents (mEPSCs), revealing that the organelle contains Ca2+ at a concentration sufficient to evoke transmitter release. Confocal laser scanning microscopy, combined with electrophysiology is used to monitor the action potential evoked increases in [Ca2+]i in boutons. We show that disruption of lysosomes compromises action potential evoked [Ca2+]i but this effect is occluded if the ER is discharged. Conversely, disruption of the lysosome does not appear to impact on the capacity of the ER to release Ca2+. These results suggest that the lysosome may serve as a Ca2+ store within hippocampal boutons, with a Ca2+ signalling role that is unique from that of the ER. PMID:16930634

McGuinness, Lindsay; Bardo, Scott J; Emptage, Nigel J

2006-08-23

246

Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content  

PubMed Central

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-?-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

Appelqvist, Hanna; Sandin, Linnea; Bjornstrom, Karin; Saftig, Paul; Garner, Brett; Ollinger, Karin; Kagedal, Katarina

2012-01-01

247

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

PubMed

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

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

2012-05-24

248

Monitoring Autophagy in Lysosomal Storage Disorders  

PubMed Central

Lysosomes are the final destination of the autophagic pathway. It is in the acidic milieu of the lysosomes that autophagic cargo is metabolized and recycled. One would expect that diseases with primary lysosomal defects would be among the first systems in which autophagy would be studied. In reality, this is not the case. Lysosomal storage diseases, a group of more than 60 diverse inherited disorders, have only recently become a focus of autophagic research. Studies of these clinically severe conditions promise not only to clarify pathogenic mechanisms, but also to expand our knowledge of autophagy itself. In this chapter, we will describe the lysosomal storage diseases in which autophagy has been explored, and present the approaches used to evaluate this essential cellular pathway.

Raben, Nina; Shea, Lauren; Hill, Victoria; Plotz, Paul

2009-01-01

249

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

PubMed Central

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.

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

250

The Adaptive Hybrid Cursor: A Pressure-Based Target Selection Technique for Pen-Based User Interfaces  

Microsoft Academic Search

We present the Adaptive Hybrid Cursor, a novel target acquisition technique for pen-based interfaces. To assist a user in a target selection task, this technique automatically adapts the size of the cursor and\\/or its contexts (the target size and the selection background) based on pen pressure input. We systematically evaluated the new technique with various 2D target acquisition tasks. The

Xiangshi Ren; Jibin Yin; Shengdong Zhao; Yang Li

2007-01-01

251

A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems  

Microsoft Academic Search

Ubiquitination generally serves as a signal for targeting cytoplasmic and nuclear proteins to the proteasome for subsequent degradation. Recently, evidence has accumulated indicating that ubiquitination also plays an important role in targeting integral membrane proteins for degradation by the lytic vacuole or the lysosome. This article describes a conserved protein motif, based on a sequence of the proteasomal component Rpn10\\/S5a,

Kay Hofmann; Laurent Falquet

2001-01-01

252

Addressing selective polypharmacology of antipsychotic drugs targeting the bioaminergic receptors through receptor dynamic conformational ensembles.  

PubMed

Selective polypharmacology, where a drug acts on multiple rather than a single molecular target involved in a disease, emerges to develop a structure-based system biology approach to design drugs selectively targeting a disease-active protein network. We focus on the bioaminergic receptors that belong to the group of G-protein-coupled receptors (GPCRs) and represent targets for therapeutic agents against schizophrenia and depression. Among them, it has been shown that the serotonin (5-HT(2A) and 5-HT?) and dopamine (D? and D?) receptors induce a cognition-enhancing effect (group 1), while the histamine (H?) and serotonin (5-HT(2C)) receptors lead to metabolic side effects and the 5-HT(2B) serotonin receptor causes pulmonary hypertension (group 2). Thus, the problem arises to develop an approach that allows identifying drugs targeting only the disease-active receptors, i.e. group 1. The recent release of several crystal structures of the bioaminergic receptors, involving the D? and H? receptors, provides the possibility to model the structures of all receptors and initiate a study of the structural and dynamic context of selective polypharmacology. In this work, we use molecular dynamics simulations to generate a conformational space of the receptors and subsequently characterize its binding properties applying molecular probe mapping. All-against-all comparison of the generated probe maps of the selected diverse conformations of all receptors with the Tanimoto similarity coefficient (Tc) enable the separation of the receptors of group 1 from group 2. The pharmacophore built based on the Tc-selected receptor conformations, using the multiple probe maps discovers structural features that can be used to design molecules selective toward the receptors of group 1. The importance of several predicted residues to ligand selectivity is supported by the available mutagenesis and ligand structure-activity relationship studies. In addition, the Tc-selected conformations of the receptors for group 1 show good performance in isolation of known ligands from a random decoy. Our computational structure-based protocol to tackle selective polypharmacology of antipsychotic drugs could be applied for other diseases involving multiple drug targets, such as oncologic and infectious disorders. PMID:23789628

Selvam, Balaji; Porter, Simon L; Tikhonova, Irina G

2013-07-05

253

Reduction of nanoparticle avidity enhances the selectivity of vascular targeting and PET detection of pulmonary inflammation.  

PubMed

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 naive 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

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

2013-02-08

254

Glioma Selectivity of Magnetically Targeted Nanoparticles: A Role of Abnormal Tumor Hydrodynamics  

PubMed Central

Magnetic targeting is a promising strategy for achieving localized drug delivery. Application of this strategy to treat brain tumors, however, is complicated by their deep intracranial location, since magnetic field density cannot be focused at a distance from an externally applied magnet. This study intended to examine whether, with magnetic targeting, pathological alteration in brain tumor flow dynamics could be of value in discriminating the diseased site from healthy brain. To address this question, the capture of magnetic nanoparticles was first assessed in vitro using a simple flow system under theoretically estimated glioma and normal brain flow conditions. Secondly, accumulation of nanoparticles via magnetic targeting was evaluated in vivo using 9L-glioma bearing rats. In vitro results that predicted a 7.6-fold increase in nanoparticle capture at glioma-versus contralateral brain-relevant flow rates were relatively consistent with the 9.6-fold glioma selectivity of nanoparticle accumulation over the contralateral brain observed in vivo. Based on these finding, the in vitro ratio of nanoparticle capture can be viewed as a plausible indicator of in vivo glioma selectivity. Overall, it can be concluded that the decreased blood flow rate in glioma, reflecting tumor vascular abnormalities, is an important contributor to glioma-selective nanoparticle accumulation with magnetic targeting.

Chertok, Beata; David, Allan E.; Huang, Yongzhuo; Yang, Victor C.

2007-01-01

255

Selection strategy to generate aptamer pairs that bind to distinct sites on protein targets.  

PubMed

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

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

2012-06-08

256

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

PubMed Central

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

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

2012-01-01

257

Biochemical differences in the mechanism of macrophage lysosomal exocytosis initiated by zymosan particles and weak bases.  

PubMed Central

By utilizing compounds with different inhibitory properties, discrete biochemical differences were found in the mechanism of selective lysosomal enzyme secretion by macrophages in response to stimulation with zymosan particles and methylamine. Pretreatment of macrophages with trypsin markedly impaired the capacity of the cells to respond to stimulation with zymosan particles, but had no effect on methylamine-stimulated lysosomal enzyme secretion. Similarly, the addition of phenylmethanesulphonyl fluoride or EDTA to the incubation medium substantially inhibited zymosan-induced lysosomal enzyme secretion, whereas the methylamine-stimulated response was unaffected by these agents. The addition of 2-deoxyglucose to incubation media, however, strongly inhibited both zymosan- and methylamine-stimulated beta-galactosidase secretion. These findings are consistent with a mechanism for lysosomal enzyme secretion by macrophages, based on a receptor-dependent uptake of zymosan particles and a receptor-independent uptake of methylamine.

Riches, D W; Watkins, J L; Stanworth, D R

1983-01-01

258

Selective Activation of Estrogen Receptor-? Target Genes by 3,3?-Diindolylmethane  

PubMed Central

3,3?-Diindolylmethane (DIM) is a natural compound found in cruciferous vegetables that has antiproliferative and estrogenic activity. However, it is not clear whether the estrogenic effects are mediated through estrogen receptor (ER)?, ER?, or both ER subtypes. We investigated whether DIM has ER subtype selectivity on gene transcription. DIM stimulated ER? but not ER? activation of an estrogen response element upstream of the luciferase reporter gene. DIM also selectively activated multiple endogenous genes through ER?. DIM did not bind to ER?, indicating that it activates genes by a ligand-independent mechanism. DIM causes ER? to bind regulatory elements and recruit the steroid receptor coactivator (SRC)-2 coactivator, which leads to the activation of ER target genes. Silencing of SRC-2 inhibited the activation of ER target genes, demonstrating that SRC-2 is required for transcriptional activation by DIM. Our results demonstrate that DIM is a new class of ER?-selective compounds, because it does not bind to ER?, but instead it selectively recruits ER? and coactivators to target genes.

Vivar, Omar I.; Saunier, Elise F.; Leitman, Dale C.; Firestone, Gary L.; Bjeldanes, Leonard F.

2010-01-01

259

Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles  

PubMed Central

Background Nanoparticle-based systems are promising for the development of imaging and therapeutic agents. The main advantage of nanoparticles over traditional systems lies in the possibility of loading multiple functionalities onto a single molecule, which are useful for therapeutic and/or diagnostic purposes. These functionalities include targeting moieties which are able to recognize receptors overexpressed by specific cells and tissues. However, targeted delivery of nanoparticles requires an accurate system design. We present here a rationally designed, genetically engineered, and chemically modified protein-based nanoplatform for cell/tissue-specific targeting. Methods Our nanoparticle constructs were based on the heavy chain of the human protein ferritin (HFt), a highly symmetrical assembly of 24 subunits enclosing a hollow cavity. HFt-based nanoparticles were produced using both genetic engineering and chemical functionalization methods to impart several functionalities, ie, the ?-melanocyte-stimulating hormone peptide as a melanoma-targeting moiety, stabilizing and HFt-masking polyethylene glycol molecules, rhodamine fluorophores, and magnetic resonance imaging agents. The constructs produced were extensively characterized by a number of physicochemical techniques, and assayed for selective melanoma-targeting in vitro and in vivo. Results Our HFt-based nanoparticle constructs functionalized with the ?-melanocyte-stimulating hormone peptide moiety and polyethylene glycol molecules were specifically taken up by melanoma cells but not by other cancer cell types in vitro. Moreover, experiments in melanoma-bearing mice indicate that these constructs have an excellent tumor-targeting profile and a long circulation time in vivo. Conclusion By masking human HFt with polyethylene glycol and targeting it with an ?-melanocyte-stimulating hormone peptide, we developed an HFt-based melanoma-targeting nanoplatform for application in melanoma diagnosis and treatment. These results could be of general interest, because the same strategy can be exploited to develop ad hoc nanoplatforms for specific delivery towards any cell/tissue type for which a suitable targeting moiety is available.

Vannucci, Luca; Falvo, Elisabetta; Fornara, Manuela; Di Micco, Patrizio; Benada, Oldrich; Krizan, Jiri; Svoboda, Jan; Hulikova-Capkova, Katarina; Morea, Veronica; Boffi, Alberto; Ceci, Pierpaolo

2012-01-01

260

Can we use genetic and genomic approaches to identify candidate animals for targeted selective treatment.  

PubMed

Estimated breeding values (EBV) for faecal egg count (FEC) and genetic markers for host resistance to nematodes may be used to identify resistant animals for selective breeding programmes. Similarly, targeted selective treatment (TST) requires the ability to identify the animals that will benefit most from anthelmintic treatment. A mathematical model was used to combine the concepts and evaluate the potential of using genetic-based methods to identify animals for a TST regime. EBVs obtained by genomic prediction were predicted to be the best determinant criterion for TST in terms of the impact on average empty body weight and average FEC, whereas pedigree-based EBVs for FEC were predicted to be marginally worse than using phenotypic FEC as a determinant criterion. Whilst each method has financial implications, if the identification of host resistance is incorporated into a wider genomic selection indices or selective breeding programmes, then genetic or genomic information may be plausibly included in TST regimes. PMID:23683653

Laurenson, Yan C S M; Kyriazakis, Ilias; Bishop, Stephen C

2013-04-26

261

Masking and triggered unmasking of targeting ligands on liposomal chemotherapy selectively suppress tumor growth in vivo.  

PubMed

We investigated the feasibility and efficacy of a drug delivery strategy to vascularized cancer that combines targeting selectivity with high uptake by targeted cells and high bioexposure of cells to delivered chemotherapeutics. Targeted lipid vesicles composed of pH responsive membranes were designed to reversibly form phase-separated lipid domains, which are utilized to tune the vesicle's apparent functionality and permeability. During circulation, vesicles mask functional ligands and stably retain their contents. Upon extravasation in the tumor interstitium, ligand-labeled lipids become unmasked and segregated within lipid domains triggering targeting to cancer cells followed by internalization. In the acidic endosome, vesicles burst release the encapsulated therapeutics through leaky boundaries around the phase-separated lipid domains. The pH tunable vesicles contain doxorubicin and are labeled with an anti-HER2 peptide. In vitro, anti-HER2 pH tunable vesicles release doxorubicin in a pH dependent manner, and exhibit 233% increase in binding to HER2-overexpressing BT474 breast cancer cells with lowering pH from 7.4 to 6.5 followed by significant (50%) internalization. In subcutaneous BT474 xenografts in nude mice, targeted pH tunable vesicles decrease tumor volumes by 159% relative to nontargeted vesicles, and they also exhibit better tumor control by 11% relative to targeted vesicles without an unmasking property. These results suggest the potential of pH tunable vesicles to ultimately control tumor growth at relatively lower administered doses. PMID:23134440

Bandekar, Amey; Zhu, Charles; Gomez, Ana; Menzenski, Monica Zofia; Sempkowski, Michelle; Sofou, Stavroula

2012-12-18

262

A galactosidase-responsive doxorubicin-folate conjugate for selective targeting of acute myelogenous leukemia blasts.  

PubMed

Cytarabine combined with an anthracycline or an anthracenedione represents the usual intensive induction therapy for the treatment of AML. However, this protocol induces severe side effects and treatment-related mortality due to the lack of selectivity of these cytotoxic agents. In this paper, we present the study of the first galactosidase-responsive molecular "Trojan Horse" programmed for the delivery of doxorubicin exclusively inside AML blasts over-expressing the folate receptor (FR). This targeting system allows the selective killing of AML blasts without affecting normal endothelial, cardiac or hematologic cells from healthy donors suggesting that FDC could reduce adverse events usually recorded with anthracyclines. PMID:23726264

Clarhaut, Jonathan; Fraineau, Sylvain; Guilhot, Joëlle; Peraudeau, Elodie; Tranoy-Opalinski, Isabelle; Thomas, Mikaël; Renoux, Brigitte; Randriamalala, Edouard; Bois, Patrick; Chatelier, Aurélien; Monvoisin, Arnaud; Cronier, Laurent; Papot, Sébastien; Guilhot, François

2013-05-28

263

The Transcription Factor TFEB Links mTORC1 Signaling to Transcriptional Control of Lysosome Homeostasis  

PubMed Central

Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity varies greatly, but the mechanisms that adjust lysosomal function to maintain cellular homeostasis are unknown. In this study, we identify an interaction between mTOR and the TFEB transcription factor on the surface of lysosomes that allows mTOR to transduce signals arising from changes in lysosomal status to TFEB and thus control the ability of TFEB to enter the nucleus. This occurs via regulation of the serine 211 phosphorylation-dependent binding of 14-3-3 proteins to TFEB. These results identify TFEB as a novel target of mTOR that couples the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. We further present evidence that the closely related MITF and TFE3 transcription factors are regulated in a similar manner, thus broadening the range of physiological contexts under which such regulation may prove important.

Roczniak-Ferguson, Agnes; Petit, Constance S.; Froehlich, Florian; Qian, Sharon; Ky, Jennifer; Angarola, Brittany; Walther, Tobias C.; Ferguson, Shawn M.

2012-01-01

264

SopE-mediated recruitment of host Rab5 on phagosomes inhibits Salmonella transport to lysosomes.  

PubMed

Phagocytosis is a process by which invading organisms are taken up by macrophages and targeted to the lysosomes, where they are degraded. However, many pathogens modulate this central process of macrophage-mediated killing by inhibiting their transport to the lysosomes through a variety of pathogen-derived mechanisms. Given the importance of Rab proteins in the regulation of intracellular transport pathways, we investigated the role of different host endocytic Rabs on the maturation of Salmonella-containing phagosomes in macrophages. Initially, we have developed a ligand mixing assay to measure the transport of the Salmonella-containing phagosomes to lysosomes. Using this assay we have shown that Salmonella decline their transport to the lysosomes. In order to determine whether inhibition of Salmonella transport to lysosomes is due to their sustained fusion with early endosomes, we have developed an in vitro fusion assay between Salmonella-containing phagosomes and early endosomes. Here, we have discussed how these methodologies are helpful to determine the mechanism of evasion of Salmonella transport to the lysosomes. PMID:18425466

Madan, Richa; Krishnamurthy, Ganga; Mukhopadhyay, Amitabha

2008-01-01

265

Sphingosine mediates TNF?-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells  

PubMed Central

Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-? (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.

Ullio, Chiara; Casas, Josefina; Brunk, Ulf T.; Sala, Giuseppina; Fabrias, Gemma; Ghidoni, Riccardo; Bonelli, Gabriella; Baccino, Francesco M.; Autelli, Riccardo

2012-01-01

266

Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights  

PubMed Central

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-? bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-? nAChR subunits.

Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B.

2009-01-01

267

Mitochondrial permeability transition pore as a selective target for anti-cancer therapy.  

PubMed

Mitochondrial outer membrane permeabilization (MOMP) is the ultimate step in dozens of lethal apoptotic signal transduction pathways which converge on mitochondria. One of the representative systems proposed to be responsible for the MOMP is the mitochondrial permeability transition pore (MPTP). Although the molecular composition of the MPTP is not clearly understood, the MPTP attracts much interest as a promising target for resolving two conundrums regarding cancer treatment: tumor selectivity and resistance to treatment. The regulation of the MPTP is closely related to metabolic reprogramming in cancer cells including mitochondrial alterations. Restoration of deregulated apoptotic machinery in cancer cells by tumor-specific modulation of the MPTP could therefore be a promising anti-cancer strategy. Currently, a number of MPTP-targeting agents are under pre-clinical and clinical studies. Here, we reviewed the structure and regulation of the MPTP as well as the current status of the development of promising MPTP-targeting drugs. PMID:23483560

Suh, Dong H; Kim, Mi-Kyung; Kim, Hee S; Chung, Hyun H; Song, Yong S

2013-03-08

268

Efficient target-selected mutagenesis in Caenorhabditis elegans: Toward a knockout for every gene  

Microsoft Academic Search

Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we implemented a target-selected knockout approach that is based on random chemical mutagenesis and detection of single nucleotide mutations in

Edwin Cuppen; Eelke Gort; Esther Hazendonk; Josine Mudde; José van de Belt; Isaäc J. Nijman; Victor Guryev; Ronald H. A. Plasterk

2007-01-01

269

A Human mAb Specific to Oncofetal Fibronectin Selectively Targets Chronic Skin Inflammation In Vivo  

Microsoft Academic Search

The antibody-based targeted delivery of bioactive agents to sites of angiogenesis is an attractive therapeutic strategy for cancer treatment, but is largely unexplored for chronic inflammatory diseases. In this article, we show that the extra domain B (EDB) domain of fibronectin, a marker of angiogenesis, is expressed in psoriatic lesions, and that the anti-EDB human antibody L19 can selectively localize

Eveline Trachsel; Manuela Kaspar; Frank Bootz; Michael Detmar; Dario Neri

2007-01-01

270

Alternative targets and economic efficiency of selecting protected areas for biodiversity conservation in boreal forest  

Microsoft Academic Search

We examine the relative merits of alternative forest biodiversity targets, which give different weights to species according\\u000a to their conservation status and abundance. A site selection framework is used for choosing the habitat-protection strategy\\u000a that maximizes biodiversity subject to an upper bound on funding with six alternative conservation goals. By using Finnish\\u000a data on old-growth forests, we found that alternative

Artti Juutinen; Mikko Mönkkönen

2007-01-01

271

TractorBeam Selection Aids: Improving Target Acquisition for Pointing Input on Tabletop Displays  

Microsoft Academic Search

This paper presents a comparison of several select ion aids to improve pointing input on tabletop displays. Our previous r esearch explored the Trac- torBeam-a hybrid point-touch interaction technique for tabletop displays. We found that while pointing input was preferred (over touch) by users of tabletop displays, it was slower for small distant targets. Drawing from previous work on improving

Karen Parker; Regan L. Mandryk; Michael N. Nunes; Kori M. Inkpen

2005-01-01

272

Adverse selection in a community-based health insurance scheme in rural Africa: Implications for introducing targeted subsidies  

PubMed Central

Background Although most community-based health insurance (CBHI) schemes are voluntary, problem of adverse selection is hardly studied. Evidence on the impact of targeted subsidies on adverse selection is completely missing. This paper investigates adverse selection in a CBHI scheme in Burkina Faso. First, we studied the change in adverse selection over a period of 4?years. Second, we studied the effect of targeted subsidies on adverse selection. Methods The study area, covering 41 villages and 1 town, was divided into 33 clusters and CBHI was randomly offered to these clusters during 2004–06. In 2007, premium subsidies were offered to the poor households. The data was collected by a household panel survey 2004–2007 from randomly selected households in these 33 clusters (n?=?6795). We applied fixed effect models. Results We found weak evidence of adverse selection before the implementation of subsidies. Adverse selection significantly increased the next year and targeted subsidies largely explained this increase. Conclusions Adverse selection is an important concern for any voluntary health insurance scheme. Targeted subsidies are often used as a tool to pursue the vision of universal coverage. At the same time targeted subsidies are also associated with increased adverse selection as found in this study. Therefore, it’s essential that targeted subsidies for poor (or other high-risk groups) must be accompanied with a sound plan to bridge the financial gap due to adverse selection so that these schemes can continue to serve these populations.

2012-01-01

273

Molecular and cellular basis of lysosomal transmembrane protein dysfunction  

Microsoft Academic Search

Lysosomal membrane proteins act at several crucial steps of the lysosome life cycle, including lumen acidification, metabolite export, molecular motor recruitment and fusion with other organelles. This review summarizes the molecular mechanisms of lysosomal storage diseases caused by defective transport of small molecules or ions across the lysosomal membrane, as well as Danon disease. In cystinosis and free sialic acid

Raquel Ruivo; Christine Anne; Corinne Sagné; Bruno Gasnier

2009-01-01

274

A Gene Network Regulating Lysosomal Biogenesis and Function  

Microsoft Academic Search

Lysosomes are organelles central to degradation and recycling processes in animal cells. Whether lysosomal activity is coordinated to respond to cellular needs remains unclear. We found that most lysosomal genes exhibit coordinated transcriptional behavior and are regulated by the transcription factor EB (TFEB). Under aberrant lysosomal storage conditions, TFEB translocated from the cytoplasm to the nucleus, resulting in the activation

Marco Sardiello; Michela Palmieri; Alberto di Ronza; Diego Luis Medina; Marta Valenza; Vincenzo Alessandro Gennarino; Chiara Di Malta; Francesca Donaudy; Valerio Embrione; Roman S. Polishchuk; Sandro Banfi; Giancarlo Parenti; Elena Cattaneo; Andrea Ballabio

2009-01-01

275

Risk-Targeted Selection of Agricultural Holdings for Post-Epidemic Surveillance: Estimation of Efficiency Gains  

PubMed Central

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

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

2011-01-01

276

Lysosome Stabilizing Agents for Hypothermic Kidney Preservation.  

National Technical Information Service (NTIS)

Chloroquine and hydrocortisone were shown to have beneficial effects on the storage of whole organs in vitro. A possible mechanism of action for these agents may be the stabilization of lysosomal membranes and the protection of the cell against autodigest...

P. A. Lotke

1966-01-01

277

Renal Lysosomal Enzyme Changes with Lithium Use.  

National Technical Information Service (NTIS)

Renal lysosomal enzyme changes with lithium use was studied. Comparisons of N-acetyl-B-glucosomidase (NAG) excretion were made in patients starting lithium, patients taking lithium for more than one year, controls, and chronic renal disordered patients. N...

M. J. Garvey

1981-01-01

278

Lysosomes and Intracellular Digestion in Sea Stars.  

National Technical Information Service (NTIS)

The study of lysosomes and intracellular digestion in sea stars seemed ideal. Echinoderms occupy an intermediate position in the phylogenetic progression from protozoan to mammal. A single sea star can cleanly provide a large amount of relatively homogeno...

G. S. Araki

1969-01-01

279

Implications of structural genomics target selection strategies: Pfam5000, whole genome, and random approaches  

SciTech Connect

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.

Chandonia, John-Marc; Brenner, Steven E.

2004-07-14

280

Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes*  

PubMed Central

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ?20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

Chen, Lishan; Zheng, Jiashun; Yang, Nan; Li, Hao; Guo, Su

2011-01-01

281

Patient selection and targeted treatment in the management of platinum-resistant ovarian cancer  

PubMed Central

Ovarian cancer (OC) has the highest mortality rate of any gynecologic cancer, and patients generally have a poor prognosis due to high chemotherapy resistance and late stage disease diagnosis. Platinum-resistant OC can be treated with cytotoxic chemotherapy such as paclitaxel, topotecan, pegylated liposomal doxorubicin, and gemcitabine, but many patients eventually relapse upon treatment. Fortunately, there are currently a number of targeted therapies in development for these patients who have shown promising results in recent clinical trials. These treatments often target the vascular endothelial growth factor pathway (eg, bevacizumab and aflibercept), DNA repair mechanisms (eg, iniparib and olaparib), or they are directed against folate related pathways (eg, pemetrexed, farletuzumab, and vintafolide). As many targeted therapies are only effective in a subset of patients, there is an increasing need for the identification of response predictive biomarkers. Selecting the right patients through biomarker screening will help tailor therapy to patients and decrease superfluous treatment to those who are biomarker negative; this approach should lead to improved clinical results and decreased toxicities. In this review the current targeted therapies used for treating platinum-resistant OC are discussed. Furthermore, use of prognostic and response predictive biomarkers to define OC patient populations that may benefit from specific targeted therapies is also highlighted.

Leamon, Christopher P; Lovejoy, Chandra D; Nguyen, Binh

2013-01-01

282

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

PubMed Central

The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of the genomic loci proximal to these binding sites introduced new candidate STAT1 and STAT2 target genes, several of which are affiliated with proliferation and apoptosis. The genes on chromosome 22 that exhibited interferon-induced up- or down-regulated expression were determined and correlated with the STAT-binding site information, revealing the potential regulatory effects of STAT1 and STAT2 on their target genes. Importantly, the comparison of STAT1-binding sites upon interferon (IFN)-? and IFN-? treatments revealed dramatic changes in binding locations between the two treatments. The IFN-? induction revealed nonconserved STAT1 occupancy at IFN-?-induced sites, as well as novel sites of STAT1 binding not evident in IFN-?-treated cells. Many of these correlated with binding by STAT2, but others were STAT2 independent, suggesting that multiple mechanisms direct STAT1 binding to its targets under different activation conditions. Overall, our results reveal a wealth of new information regarding IFN/STAT-binding targets and also fundamental insights into mechanisms of regulation of gene expression in different cell states.

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

2005-01-01

283

Involvement of BimL activation in apoptosis induced by lysosomal photodamage  

NASA Astrophysics Data System (ADS)

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

Liu, Lei; Wang, Xianwang; Li, Hui

2008-12-01

284

Membrane Resealing Mediated by Lysosomal Exocytosis  

Microsoft Academic Search

Ca2+-regulated exocytosis was proposed to mediate plasma membrane repair, but until recently the nature of the vesicles involved\\u000a was unknown. Recent work from our laboratory identified lysosomes as the intracellular organelles responsible for this process.\\u000a Ca2+ triggers the exocytosis of conventional lysosomes, and this process is regulated by a ubiquitously expressed synaptotagmin,\\u000a Syt VII. Dominant-negative or gene deletion approaches revealed

Norma W. Andrews

285

Lysosomal Cysteine Proteases and Antigen Presentation  

Microsoft Academic Search

Lysosomal proteinases are involved in two critical stages of MHC class II-mediated antigen presentation, i.e., degradation\\u000a of invariant chain, a chaperone molecule critical for MHC class II assembly and transport, and generation of class II-binding\\u000a peptides in the endocytic compartment. We found that two lysosomal cysteine proteinases, cathepsins S and L, were found to\\u000a be differentially expressed in different types

A. Rudensky; C. Beers

286

Lysosomal cysteine proteases regulate antigen presentation  

Microsoft Academic Search

Antigen presentation by both classical MHC class II molecules and the non-classical MHC class I-like molecule CD1D requires their entry into the endosomal\\/lysosomal compartment. Lysosomal cysteine proteases constitute an important subset of the enzymes that are present in this compartment and, here, we discuss the role of these proteases in regulating antigen presentation by both MHC class II and CD1D

Alexander Y. Rudensky; Karen Honey

2003-01-01

287

Multiple-Targeting and Conformational Selection in the Estrogen Receptor: Computation and Experiment  

PubMed Central

Conformational selection is a primary mechanism in biomolecular recognition. The conformational ensemble may determine the ability of a drug to compete with a native ligand for a receptor target. Traditional docking procedures which use one or few protein structures are limited and may not be able to represent a complex competition among closely related protein receptors in agonist and antagonist ensembles. Here, we test a protocol aimed at selecting a drug candidate based on its ability to synergistically bind to distinct conformational states. We demonstrate, for the case of estrogen receptor ? (ER?) and estrogen receptor ? (ER?), that the functional outcome of ligand binding can be inferred from its ability to simultaneously bind both ER? and ER? in agonist and antagonist conformations as calculated docking scores. Combining a conformational selection method with an experimental reporter gene system in yeast, we propose that several phytoestrogens can be novel estrogen receptor ? selective agonists. Our work proposes a computational protocol to select estrogen receptor subtype selective agonists. Compared with other models, present method gives the best prediction in ligands’ function.

Yuan, Peng; Liang, Kaiwei; Ma, Buyong; Zheng, Nan; Nussinov, Ruth; Huang, Jian

2011-01-01

288

Crohn's disease loci are common targets of protozoa-driven selection.  

PubMed

Previous studies indicated that a few risk variants for autoimmune diseases are subject to pathogen-driven selection. Nonetheless, the proportion of risk loci that has been targeted by pathogens and the type of infectious agent(s) that exerted the strongest pressure remain to be evaluated. We assessed whether different pathogens exerted a pressure on known Crohn's disease (CD) risk variants and demonstrate that these single-nucleotide polymorphisms (SNPs) are preferential targets of protozoa-driven selection (P = 0.008). In particular, 19% of SNPs associated with CD have been subject to protozoa-driven selective pressure. Analysis of P values from genome-wide association studies (GWASs) and meta-analyses indicated that protozoan-selected SNPs display significantly stronger association with CD compared with nonselected variants. This same behavior was not observed for GWASs of other autoimmune diseases. Thus, we integrated selection signatures and meta-analysis results to prioritize five genic SNPs for replication in an Italian cohort. Three SNPs were significantly associated with CD risk, and combination with meta-analysis results yielded P values < 4 × 10(-6). The bona fide risk alleles are located in ARHGEF2, an interactor of NOD2, NSF, a gene involved in autophagy, and HEBP1, encoding a possible mediator of inflammation. Pathway analysis indicated that ARHGEF2 and NSF participate in a molecular network, which also contains VAMP3 (previously associated to CD) and is centered around miR-31 (known to be disregulated in CD). Thus, we show that protozoa-driven selective pressure had a major role in shaping predisposition to CD. We next used this information for the identification of three bona fide novel susceptibility loci. PMID:23389767

Cagliani, Rachele; Pozzoli, Uberto; Forni, Diego; Cassinotti, Andrea; Fumagalli, Matteo; Giani, Matteo; Fichera, Maria; Lombardini, Marta; Ardizzone, Sandro; Asselta, Rosanna; de Franchis, Roberto; Riva, Stefania; Biasin, Mara; Comi, Giacomo P; Bresolin, Nereo; Clerici, Mario; Sironi, Manuela

2013-02-06

289

Lysosomal Storage Disorders in the Newborn  

PubMed Central

Lysosomal storage disorders are rare inborn errors of metabolism, with a combined incidence of 1 in 1500 to 7000 live births. These relatively rare disorders are seldom considered when evaluating a sick newborn. A significant number of the >50 different lysosomal storage disorders, however, do manifest in the neonatal period and should be part of the differential diagnosis of several perinatal phenotypes. We review the earliest clinical features, diagnostic tests, and treatment options for lysosomal storage disorders that can present in the newborn. Although many of the lysosomal storage disorders are characterized by a range in phenotypes, the focus of this review is on the specific symptoms and clinical findings that present in the perinatal period, including neurologic, respiratory, endocrine, and cardiovascular manifestations, dysmorphic features, hepatosplenomegaly, skin or ocular involvement, and hydrops fetalis/congenital ascites. A greater awareness of these features may help to reduce misdiagnosis and promote the early detection of lysosomal storage disorders. Implementing therapy at the earliest stage possible is crucial for several of the lysosomal storage disorders; hence, an early appreciation of these disorders by physicians who treat newborns is essential.

Staretz-Chacham, Orna; Lang, Tess C.; LaMarca, Mary E.; Krasnewich, Donna; Sidransky, Ellen

2009-01-01

290

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

PubMed Central

Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1–V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacer and Enterococcus predominated in the community generated by V4–V6 primers, and the most numerous genera in the V7–V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4–V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7–V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1–V3 and V7–V9 primers providesd results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.

Kumar, Purnima S.; Brooker, Michael R.; Dowd, Scot E.; Camerlengo, Terry

2011-01-01

291

Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by CoTransformation and Anther Culture  

Microsoft Academic Search

The ‘double T-DNA’ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene (hpt) in one T-DNA region and three target genes (hLF, SB401, RZ10) in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for

Li ZHU; Ya-ping FU; Wen-zhen LIU; Guo-cheng HU; Hua-min SI; Ke-xuan TANG; Zong-xiu SUN

2007-01-01

292

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

NASA Astrophysics Data System (ADS)

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

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

2009-06-01

293

Rheb and Rags come together at the lysosome to activate mTORC1.  

PubMed

mTORC1 (mammalian target of rampamycin complex 1) is a highly conserved protein complex regulating cell growth and metabolism via its kinase mTOR (mammalian target of rapamycin). The activity of mTOR is under the control of various GTPases, of which Rheb and the Rags play a central role. The presence of amino acids is a strict requirement for mTORC1 activity. The heterodimeric Rag GTPases localize mTORC1 to lysosomes by their amino-acid-dependent interaction with the lysosomal Ragulator complex. Rheb is also thought to reside on lysosomes to activate mTORC1. Rheb is responsive to growth factors, but, in conjunction with PLD1 (phospholipase D1), is also an integral part of the machinery that stimulates mTORC1 in response to amino acids. In the present article, we provide a brief overview of novel mechanisms by which amino acids affect the function of Rags. On the basis of existing literature, we postulate that Rheb is activated at the Golgi from where it will travel to lysosomes. Maturation of endosomes into lysosomes may be required to assure a continuous supply of GTP-bound Rheb for mTORC1 activation, which may help to drive the maturation process. PMID:23863162

Groenewoud, Marlous J; Zwartkruis, Fried J T

2013-08-01

294

Selective Targeting of RANK Signaling Pathways as New Therapeutic Strategies for Osteoporosis  

PubMed Central

Importance of the field Osteoporosis has become a worldwide health and social issue due to an aging population. Four major antiresorptive drugs (agents capable of inhibiting osteoclast formation and/or function) are currently available on the market: estrogen, selective estrogen receptor modulators (SERMs), bisphosphonates and calcitonin. These drugs either lack satisfactory efficacy or have potential to cause serious side effects. Thus, development of more efficacious and safer drugs is warranted. Areas covered in this review The discovery of the receptor activator of NF-?B Ligand (RANKL) and its two receptors, RANK and osteoprotegerin (OPG), has not only established a crucial role for the RANKL/RANK/OPG axis in osteoclast biology but also created a great opportunity to develop new drugs targeting this system for osteoporosis therapy. This review focuses on discussion of therapeutic targeting of RANK signaling. What the reader will gain An update on the functions of RANKL and an overview of the known RANK signaling pathways in osteoclasts. A discussion of rationales for exploring RANK signaling pathways as potent and specific therapeutic targets to promote future development of better drugs for osteoporosis. Take home message Several RANK signaling components have the potential to serve as potent and specific therapeutic targets for osteoporosis.

Jules, Joel; Ashley, Jason W.; Feng, Xu

2010-01-01

295

Interactions between autophagic and endo-lysosomal markers in endothelial cells.  

PubMed

Autophagic and endo-lysosomal degradative pathways are essential for cell homeostasis. Availability of reliable tools to interrogate these pathways is critical to unveil their involvement in physiology and pathophysiology. Although several probes have been recently developed to monitor autophagic or lysosomal compartments, their specificity has not been validated through co-localization studies with well-known markers. Here, we evaluate the selectivity and interactions between one lysosomal (Lyso-ID) and one autophagosomal (Cyto-ID) probe under conditions modulating autophagy and/or endo-lysosomal function in live cells. The probe for acidic compartments Lyso-ID was fully localized inside vesicles positive for markers of late endosome-lysosomes, including Lamp1-GFP and GFP-CINCCKVL. Induction of autophagy by amino acid deprivation in bovine aortic endothelial cells caused an early and potent increase in the fluorescence of the proposed autophagy dye Cyto-ID. Cyto-ID-positive compartments extensively co-localized with the autophagosomal fluorescent reporter RFP-LC3, although the time and/or threshold for organelle detection was different for each probe. Interestingly, use of Cyto-ID in combination with Lysotracker Red or Lyso-ID allowed the observation of structures labeled with either one or both probes, the extent of co-localization increasing upon treatment with protease inhibitors. Inhibition of the endo-lysosomal pathway with chloroquine or U18666A resulted in the formation of large Cyto-ID and Lyso-ID-positive compartments. These results constitute the first assessment of the selectivity of Cyto-ID and Lyso-ID as probes for the autophagic and lysosomal pathways, respectively. Our observations show that these probes can be used in combination with protein-based markers for monitoring the interactions of both pathways in live cells. PMID:23203316

Oeste, Clara L; Seco, Esther; Patton, Wayne F; Boya, Patricia; Pérez-Sala, Dolores

2012-12-01

296

Candidate Targets of Balancing Selection in the Genome of Staphylococcus aureus  

PubMed Central

Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajima's D (>2.03) and ?/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen.

Thomas, Jonathan C.; Godfrey, Paul A.; Feldgarden, Michael; Robinson, D. Ashley

2012-01-01

297

DRAM1 Regulates Autophagy Flux through Lysosomes  

PubMed Central

We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes.

Wu, Jun-Chao; Qin, Zheng-Hong

2013-01-01

298

DRAM1 regulates autophagy flux through lysosomes.  

PubMed

We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes. PMID:23696801

Zhang, Xing-Ding; Qi, Lin; Wu, Jun-Chao; Qin, Zheng-Hong

2013-05-17

299

Correlation between OFF and ON channels underlies dark target selectivity in an insect visual system.  

PubMed

In both vertebrates and invertebrates, evidence supports separation of luminance increments and decrements (ON and OFF channels) in early stages of visual processing (Hartline, 1938; Joesch et al., 2010); however, less is known about how these parallel pathways are recombined to encode form and motion. In Drosophila, genetic knockdown of inputs to putative ON and OFF pathways and direct recording from downstream neurons in the wide-field motion pathway reveal that local elementary motion detectors exist in pairs that separately correlate contrast polarity channels, ON with ON and OFF with OFF (Joesch et al., 2013). However, behavioral responses to reverse-phi motion of discrete features reveal additional correlations of the opposite signs (Clark et al., 2011). We here present intracellular recordings from feature detecting neurons in the dragonfly that provide direct physiological evidence for the correlation of OFF and ON pathways. These neurons show clear polarity selectivity for feature contrast, responding strongly to targets that are darker than the background and only weakly to dark contrasting edges. These dark target responses are much stronger than the linear combination of responses to ON and OFF edges. We compare these data with output from elementary motion detector-based models (Eichner et al., 2011; Clark et al., 2011), with and without stages of strong center-surround antagonism. Our data support an alternative elementary small target motion detector model, which derives dark target selectivity from the correlation of a delayed OFF with an un-delayed ON signal at each individual visual processing unit (Wiederman et al., 2008, 2009). PMID:23926274

Wiederman, Steven D; Shoemaker, Patrick A; O'Carroll, David C

2013-08-01

300

PIF-Pocket as a Target for C. albicans Pkh Selective Inhibitors.  

PubMed

The phosphoinositide-dependent protein kinase 1, PDK1, is a master kinase that phosphorylates the activation loop of up to 23 AGC kinases. S. cerevisiae has three PDK1 orthologues, Pkh1-3, which also phosphorylate AGC kinases (e.g., Ypk, Tpk, Pkc1, and Sch9). Pkh1 and 2 are redundant proteins involved in multiple essential cellular functions, including endocytosis and cell wall integrity. Based on similarities with the budding yeast, the Pkh of fungal infectious species was postulated as a novel target for antifungals. Here, we found that depletion of Pkh eventually induces oxidative stress and DNA double-strand breaks, leading to programmed cell death. This finding supports Pkh as an antifungal target since pharmacological inhibition of Pkh would lead to the death of yeast cells, the ultimate goal of antifungals. It was therefore of interest to further investigate the possibility to develop Pkh inhibitors with selectivity for Candida Pkh that would not inhibit the human ortholog. Here, we describe C. albicans Pkh2 biochemically, structurally and by using chemical probes in comparison to human PDK1. We found that a regulatory site on the C. albicans Pkh2 catalytic domain, the PIF-pocket, diverges from human PDK1. Indeed, we identified and characterized PS77, a new small allosteric inhibitor directed to the PIF-pocket, which has increased selectivity for C. albicans Pkh2. Together, our results describe novel features of the biology of Pkh and chemical biology approaches that support the validation of Pkh as a drug target for selective antifungals. PMID:23911092

Pastor-Flores, Daniel; Schulze, Jörg O; Bahí, Anna; Giacometti, Romina; Ferrer-Dalmau, Jofre; Passeron, Susana; Engel, Matthias; Süß, Evelyn; Casamayor, Antonio; Biondi, Ricardo M

2013-08-22

301

Transcriptional activation of lysosomal exocytosis promotes cellular clearance.  

PubMed

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

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

2011-09-01

302

Lysosomal delivery of therapeutic enzymes in cell models of Fabry disease.  

PubMed

The success of enzymatic replacement in Gaucher disease has stimulated development of targeted protein replacement for other lysosomal disorders, including Anderson-Fabry disease, which causes fatal cardiac, cerebrovascular and renal injury: deficiency of lysosomal ?-Galactosidase A induces accumulation of glycosphingolipids. Endothelial cell storage was the primary endpoint in a clinical trial that led to market authorization. Two ?-Galactosidase A preparations are licensed worldwide, but fatal outcomes persist, with storage remaining in many tissues. We compare mechanisms of uptake of ? -Galactosidase A into cells relevant to Fabry disease, in order to investigate if the enzyme is targeted to the lysosomes in a mannose-6-phosphate receptor dependent fashion, as generally believed. ? -Galactosidase A uptake was examined in fibroblasts, four different endothelial cell models, and hepatic cells in vitro. Uptake of europium-labeled human ? -Galactosidase A was measured by time-resolved fluorescence. Ligand-specific uptake was quantified in inhibitor studies. Targeting to the lysosome was determined by precipitation and by confocal microscopy. The quantity and location of cation-independent mannose-6-phosphate receptors in the different cell models were investigated using confocal microscopy. Uptake and delivery of ? -Galactosidase A to lysosomes in fibroblasts is mediated by the canonical mannose-6-phosphate receptor pathway, but in endothelial cells in vitro this mechanism does not operate. Moreover, this observation is supported by a striking paucity of expression of cation independent mannose-6-phosphate receptors on the plasma membrane of the four endothelial cell models and by little delivery of enzyme to lysosomes, when compared with fibroblasts. If these observations are confirmed in vivo, alternative mechanisms will be needed to explain the ready clearance of storage from endothelial cells in patients undergoing enzyme replacement therapy. PMID:22450713

Marchesan, D; Cox, T M; Deegan, P B

2012-03-24

303

Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing  

PubMed Central

We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21?408 CGIs and more than 15?946 transcriptional regulatory regions. Of the CpGs analyzed, 77–84% fell on or near capture probe sequences; 69–75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5?-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.

Lee, Eun-Joon; Pei, Lirong; Srivastava, Gyan; Joshi, Trupti; Kushwaha, Garima; Choi, Jeong-Hyeon; Robertson, Keith D.; Wang, Xinguo; Colbourne, John K.; Zhang, Lu; Schroth, Gary P.; Xu, Dong; Zhang, Kun; Shi, Huidong

2011-01-01

304

Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes.  

PubMed

In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types. PMID:15090140

Nomanbhoy, Tyzoon K; Rosenblum, Jonathan; Aban, Arwin; Burbaum, Jonathan J

2003-02-01

305

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

SciTech Connect

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.

Gorria, Morgane; Tekpli, Xavier; Rissel, Mary [Inserm U620, Group Toxicity of polycyclic aromatic hydrocarbons, Equipe Labellisee Ligue contre le Cancer, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France)]|[Universite Rennes 1, IFR140, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France); Sergent, Odile [UPRES EA 3891, UFR des Sciences Pharmaceutiques et Biologiques, Universite de Rennes 1, 2, av. Pr. Leon Bernard, 34043 Rennes cedex (France); Huc, Laurence [Inserm U620, Group Toxicity of polycyclic aromatic hydrocarbons, Equipe Labellisee Ligue contre le Cancer, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France)]|[Universite Rennes 1, IFR140, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France); Landvik, Nina [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Fardel, Olivier; Dimanche-Boitrel, Marie-Therese [Inserm U620, Group Toxicity of polycyclic aromatic hydrocarbons, Equipe Labellisee Ligue contre le Cancer, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France)]|[Universite Rennes 1, IFR140, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France); Holme, Jorn A. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Lagadic-Gossmann, Dominique [Inserm U620, Group Toxicity of polycyclic aromatic hydrocarbons, Equipe Labellisee Ligue contre le Cancer, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France)]|[Universite Rennes 1, IFR140, 2 av Pr. Leon Bernard, 35043 Rennes cedex (France)], E-mail: dominique.lagadic@rennes.inserm.fr

2008-04-15

306

In situ selection of lead compounds by click chemistry: target-guided optimization of acetylcholinesterase inhibitors.  

PubMed

The target-guided, in situ click chemistry approach to lead discovery has been successfully employed for discovering acetylcholinesterase (AChE) inhibitors by incubating a selected enzyme/tacrine azide combination with a variety of acetylene reagents that were not previously known to interact with the enzyme's peripheral binding site. The triazole products, formed by the enzyme, were identified by HPLC-mass spectrometry analysis of the crude reaction mixtures. The target-guided lead discovery search was also successful when performed with reagent mixtures containing up to 10 components. From 23 acetylene reagents, the enzyme selected two phenyltetrahydroisoquinoline (PIQ) building blocks that combined with the tacrine azide within the active center gorge to form multivalent inhibitors that simultaneously associate with the active and peripheral binding sites. These new inhibitors are up to 3 times as potent as our previous phenylphenanthridinium-derived compounds, and with dissociation constants as low as 33 femtomolar, they are the most potent noncovalent AChE inhibitors known. In addition, the new compounds lack a permanent positive charge and aniline groups and possess fewer fused aromatic rings. Remarkably, despite the high binding affinity, the enzyme displayed a surprisingly low preference for one PIQ enantiomer over the other. PMID:15869290

Krasi?ski, Antoni; Radi?, Zoran; Manetsch, Roman; Raushel, Jessica; Taylor, Palmer; Sharpless, K Barry; Kolb, Hartmuth C

2005-05-11

307

Update on the Pfam5000 Strategy for Selection of StructuralGenomics Targets  

SciTech Connect

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.

Chandonia, John-Marc; Brenner, Steven E.

2005-06-27

308

Sejong Open Cluster Survey (SOS). 0. Target Selection and Data Analysis  

NASA Astrophysics Data System (ADS)

Star clusters are superb astrophysical laboratories containing cospatial and coeval samples of stars with similar chemical composition. We initiate the Sejong Open cluster Survey (SOS) - a project dedicated to providing homogeneous photometry of a large number of open clusters in the SAAO Johnson-Cousins' UBVI system. To achieve our main goal, we pay much attention to the observation of standard stars in order to reproduce the SAAO standard system. Many of our targets are relatively small sparse clusters that escaped previous observations. As clusters are considered building blocks of the Galactic disk, their physical properties such as the initial mass function, the pattern of mass segregation, etc. give valuable information on the formation and evolution of the Galactic disk. The spatial distribution of young open clusters will be used to revise the local spiral arm structure of the Galaxy. In addition, the homogeneous data can also be used to test stellar evolutionary theory, especially concerning rare massive stars. In this paper we present the target selection criteria, the observational strategy for accurate photometry, and the adopted calibrations for data analysis such as color-color relations, zero-age main sequence relations, Sp - M_V relations, Sp - T_{eff} relations, Sp - color relations, and T_{eff} - BC relations. Finally we provide some data analysis such as the determination of the reddening law, the membership selection criteria, and distance determination.

Sung, Hwankyung; Lim, Beomdu; Bessell, Michael S.; Kim, Jinyoung S.; Hur, Hyeonoh; Chun, Moo-Young; Park, Byeong-Gon

2013-06-01

309

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics  

PubMed Central

Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications (PTMs), as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.

Shi, Tujin; Su, Dian; Liu, Tao; Tang, Keqi; Camp, David G.; Qian, Wei-Jun; Smith, Richard D.

2012-01-01

310

Selecting Targeted Symptoms/Syndromes for Syndromic Surveillance in Rural China  

PubMed Central

Objective To select the potential targeted symptoms/syndromes as early warning indicators for epidemics or outbreaks detection in rural China. Introduction Patients’ chief complaints (CCs) as a common data source, has been widely used in syndromic surveillance due to its timeliness, accuracy and availability (1). For automated syndromic surveillance, CCs always classified into predefined syndromic categories to facilitate subsequent data aggregation and analysis. However, in rural China, most outpatient doctors recorded the information of patients (e.g. CCs) into clinic logs manually rather than computers. Thus, more convenient surveillance method is needed in the syndromic surveillance project (ISSC). And the first and important thing is to select the targeted symptoms/syndromes. Methods Epidemiological analysis was conducted on data from case report system in Jingmen City (one study site in ISSC) from 2004 to 2009. Initial symptoms/syndromes were selected by literature reviews. And finally expert consultation meetings, workshops and field investigation were held to confirm the targeted symptoms/syndromes. Results 10 kinds of infectious diseases, 6 categories of emergencies, and 4 bioterrorism events (i.e. plague, anthrax, botulism and hemorrhagic fever) were chose as specific diseases/events for monitoring (Table 1). Two surveillance schemes were developed by reviewing on 565 literatures about clinical conditions of specific diseases/events and 14 literatures about CCs based syndromic surveillance. The former one was to monitor symptoms (19 initial symptoms), and then aggregation or analysis on single or combined symptom(s); and the other one was to monitor syndromes (9 initial syndromes) directly (Table 2). The consultation meeting and field investigation identified three issues which should be considered: 1) the abilities of doctors especially village doctors to understand the definitions of symptoms/syndromes; 2) the workload of data collection; 3) the sensitive and specific of each symptom/syndrome. Finally, Scheme 1 was used and 10 targeted symptoms were determined (Table 2). Conclusions We should take the simple, stability and feasibility of operation, and also the local conditions into account before establishing a surveillance system. Symptoms were more suitable for monitoring compared to syndromes in resource-poor settings. Further evaluated and validated would be conducted during implementation. Our study might provide methods and evidences for other developing countries with limited conditions in using automated syndromic surveillance system, to construct similar early warning system.

Tan, Li; Zhang, Jie; Cheng, Liwei; Yan, Weirong; Diwan, Vinod K.; Long, Lu; Nie, Shaofa

2013-01-01

311

ADAM17-overexpressing breast cancer cells selectively targeted by antibody-toxin conjugates.  

PubMed

A disintegrin and metalloproteinase 17 (ADAM17) is significantly upregulated not only in malignant cells but also in the pro-inflammatory microenvironment of breast cancer. There, ADAM17 is critically involved in the processing of tumor-promoting proteins. Therefore, ADAM17 appears to be an attractive therapeutic target to address not only tumor cells but also the tumor-promoting environment. In a previous study, we generated a monoclonal anti-ADAM17 antibody (A300E). Although showing no complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity, the antibody was rapidly internalized by ADAM17-expressing cells and was able to transport a conjugated toxin into target cells. As a result, doxorubicin-coupled A300E or Pseudomonas exotoxin A-loaded A300E was able to kill ADAM17-expressing cells. This effect was strictly dependent on the presence of ADAM17 on the surface of target cells. As a proof of principle, both immunotoxins killed MDA-MB-231 breast cancer cells in an ADAM17-dependent manner. These data suggest that the use of anti-ADAM17 monoclonal antibodies as a carrier might be a promising new strategy for selective anti-cancer drug delivery. PMID:22940887

Trad, Ahmad; Hansen, Hinrich P; Shomali, Mohammad; Peipp, Matthias; Klausz, Katja; Hedemann, Nina; Yamamoto, Kosuke; Mauermann, André; Desel, Christine; Lorenzen, Inken; Lemke, Hilmar; Rose-John, Stefan; Grötzinger, Joachim

2012-09-01

312

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

PubMed Central

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.

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

313

Target detection as a tool of selective spray application on trees and weeds in orchards  

NASA Astrophysics Data System (ADS)

A spectral detection system discriminating the targets for the non target area was tested during spray applications in apple and pear orchards. The objective of the test was to evaluate the accuracy of the system working at different application parameters and to estimate the rate of possible spray savings obtained during applications on the trees of different size and weeds of different density. The system consisted of the spray units equipped with optic sensor and a control unit which could operate up to 16 spray units. Each spray unit had an optic detector and two light sources emitting two beams of light at the wavelengths 670 and 750. The ratio between emitted and reflected light for each wavelength was the basis for discriminating between the presence or the absence of chlorophyll. The information was processed and used to control the electric solenoid valves opening or shutting off the nozzles. The target detection system worked technically properly. It enabled the selective spray application with spray savings adequate to the tree row profile. In intensive apple and pear orchards 16-25 percent reduction of spray volume was obtained. For herbicide applications the detection system discriminated weeds for the bare ground. Both sensitivity of the sensors and weed density had a significant influence on the spray savings. At medium sensitivity, a considerable spray saving amounting 23 percent was obtained only on the plots with very low weed coverage.

Doruchowski, Grzegorz; Jaeken, Peter; Holownicki, Ryszard

1999-01-01

314

Selective targeting of distinct active site nucleophiles by irreversible SRC-family kinase inhibitors.  

PubMed

Src-family tyrosine kinases play pivotal roles in human physiology and disease, and several drugs that target members of this family are in clinical use. None of these drugs appear to discriminate among closely related kinases. However, assessing their selectivity toward endogenous kinases in living cells remains a significant challenge. Here, we report the design of two Src-directed chemical probes, each consisting of a nucleoside scaffold with a 5'-electrophile. A 5'-fluorosulfonylbenzoate (1) reacts with the conserved catalytic lysine (Lys295) and shows little discrimination among related kinases. By contrast, a 5'-vinylsulfonate (2) reacts with a poorly conserved, proximal cysteine (Cys277) found in three Src-family and six unrelated kinases. Both 1 and 2 bear an alkyne tag and efficiently label their respective endogenous kinase targets in intact cells. Using 1 as a competitive probe, we determined the extent to which ponatinib, a clinical Bcr-Abl inhibitor, targets Src-family kinases. Remarkably, while ponatinib had little effect on endogenous Fyn or Src, it potently blocked the critical T-cell kinase, Lck. Probes 1 and 2 thus enable competitive profiling versus distinct kinase subsets in living cells. PMID:23190395

Gushwa, Nathan N; Kang, Sumin; Chen, Jing; Taunton, Jack

2012-12-04

315

Attaching the phage display-selected GLA peptide to liposomes: factors influencing target binding.  

PubMed

In our previous study, phage display selections were performed by in situ perfusion of a random peptide library through a mouse brain. This yielded two peptides (GLA and GYR) that showed significant binding to human brain endothelial cells (hCMEC/D3) when displayed on phage particles, but not to human umbilical vein endothelial cells (HUVECs). In the present study, these peptides were produced synthetically and coupled to liposomes to investigate the capacity of the peptides to act as ligands for targeting to hCMEC/D3 cells. Flow cytometry studies showed that these peptides when coupled to liposomes showed weak binding to the target brain endothelial cells. We hypothesized that the weak endothelial cell binding of the selected peptides when coupled to liposomes as compared to the binding of the peptides displayed on phage particles may be ascribed to: change of vehicle shape, change of peptide density, or change of peptide conformation. Peptide density on the liposomes influenced binding of the liposomes to the cells, however, this effect was minor. To study the influence of the peptide conformation, the GLA peptide was recombinantly produced fused to the N1-N2 domains of the phage p3 minor coat protein (p3-GLA) to mimic its conformation when displayed on phage particles. Binding of liposomes modified with either the GLA peptide or the p3-GLA protein to hCMEC/D3 cells was studied, and the p3-GLA-liposomes showed a higher binding to the cells compared to the GLA-liposomes. The experiments demonstrate that bringing the GLA peptide into the original phage protein environment restores and improves the peptide binding capacity and suggest that the GLA peptide, with some modifications, may be used as a brain-targeting ligand in the future. PMID:22155541

van Rooy, Inge; Hennink, Wim E; Storm, Gert; Schiffelers, Raymond M; Mastrobattista, Enrico

2011-12-03

316

Analysis of the mosquito lysosomal asparticp protease gene: An insect housekeeping gene with fat body-enhanced expression  

Microsoft Academic Search

In the fat body of insects with cyclic egg maturation, lysosomes play a critical role in the termination of vitellogenesis by selectively degrading the secretory machinery involved in the massive production of yolk protein precursors. To investigate this fat body-specific lysosomal activity in the mosquito, a cathepsin D-like aspartic protease (LAP) was previously purified and its cDNA cloned. Here we

Neal T. Dittmer; Alexander S. Raikhel

1997-01-01

317

Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages.  

PubMed

Moderate lysosomal membrane permeabilization (LMP) is an important inducer of apoptosis. Macrophages are professional scavengers and are rich in hydrolytic enzymes and iron. In the present study, we found that LMP by lysosomotropic detergent MSDH resulted in early up-regulation of lysosomal cathepsins, oxidative stress and ferritin up-regulation, and cell death. Lysosomotropic base NH(4)Cl reduced the ferritin induction and oxidative stress in apoptotic cells induced by MSDH. Cysteine cathepsin inhibitors significantly protected cell death and oxidative stress, but had less effect on ferritin induction. We conclude that oxidative stress induced by lysosomal rupture causes ferritin induction with concomitant mitochondrial damage, which are the potential target for prevention of cellular oxidative stress and cell death induced by typical lysosomotropic substances in different disorders. PMID:21219901

Ghosh, Moumita; Carlsson, Fredrik; Laskar, Amit; Yuan, Xi-Ming; Li, Wei

2011-01-08

318

Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor.  

PubMed

Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of unknown physiological function. We now demonstrate that this protease activity triggers the degradation of the lysosome-associated membrane protein type 2a (lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of lamp2a is important because its level in the lysosomal membrane is a rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a degradation, higher levels of lamp2a and higher rates of CMA. Restoration of PPCA protease activity increases rates of lamp2a degradation, reduces levels of lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the lysosomal membrane and cleaves lamp2a near the boundary between the luminal and transmembrane domains. In addition to the well-studied role of PPCA in targeting and protecting two lysosomal glycosidases, we have defined a role for the proteolytic activity of this multifunctional protein. PMID:12505983

Cuervo, Ana Maria; Mann, Linda; Bonten, Erik J; d'Azzo, Alessandra; Dice, J Fred

2003-01-01

319

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

PubMed

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

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

2013-08-27

320

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

PubMed

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

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

2011-10-05

321

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

Microsoft Academic Search

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, H3 receptor, 5-HT1A receptor, 5-HT1B receptor,

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

322

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Protein-induced Lysosomal Translocation of Proapoptotic Effectors Is Mediated by Phosphofurin Acidic Cluster Sorting Protein-2 (PACS-2)*  

PubMed Central

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.

Werneburg, Nathan W.; Bronk, Steve F.; Guicciardi, Maria Eugenia; Thomas, Laurel; Dikeakos, Jimmy D.; Thomas, Gary; Gores, Gregory J.

2012-01-01

323

Arylsulfatase k, a novel lysosomal sulfatase.  

PubMed

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

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

2013-08-28

324

Recruitment of OKM1 staining lymphocytes with selective binding to K-562 tumour targets by interferon.  

PubMed Central

Spontaneous cytotoxicity of human lymphocytes for tumours is increased by interferon (IFN) without change in the overall fraction of cells binding to targets. We developed an indirect immunofluorescent technique to stain lymphocytes conjugated to K-562 tumour cells in agarose with monoclonal antibodies. This allowed assessment of lymphocyte subpopulations binding to tumour cells without disruption of conjugates. Overall binding of non-adherent (NA) lymphocytes to tumour targets following incubation at 37 degrees C for 6 h was 13.3 +/- 0.3% compared to 12.5 +/- 0.7% with inclusion of IFN at 100 u/ml. When NA lymphocytes were incubated with K-562 tumour cells without IFN, OKM1 and OKT3 staining lymphocytes comprised 16.8 +/- 3.5% and 83.0 +/- 1.3% of the total lymphocyte population and 32.5 +/- 1.3% and 70.2 +/- 2.6% of lymphocytes conjugated to tumours. Incubation with IFN significantly increased OKM1 staining cells in the total NA population to 57.2 +/- 5.6% (P less than 0.01) and within tumour conjugates to 59.2 +/- 2.7% (P less than 0.01) while OKT3 staining cells decreased to 58.3 +/- 5.2% (P less than 0.02) and 45.3 +/- 1.2% (P less than 0.01), respectively. IFN increased cytotoxicity of NA cells for 51Cr-labelled K-562 by 66% at an effector to target ratio of 30:1 (P less than 0.001). These results demonstrate that OKM1 staining cells bind more avidly to tumour targets in the absence of IFN. IFN selectively increases the proportion of OKM1 staining lymphocytes with a concomitant increase in their binding to tumour cells. Therefore, enhancement of cytotoxicity by IFN in the NK system may result, in part, from conversion of OKT3 to OKM1 staining cells which are more efficient killers.

Salata, R A; Schacter, B Z; Ellner, J J

1983-01-01

325

Identification of Lysosomal and Golgi Localization Signals in GAP and ARF Domains of ARF Domain Protein 1  

PubMed Central

ADP ribosylation factors (ARFs) are ?20-kDa guanine nucleotide-binding proteins that activate cholera toxin and phospholipase D and are critical components of vesicular trafficking pathways. ARF domain protein 1 (ARD1), a member of the ARF superfamily, contains a 46-kDa amino-terminal extension, which acts as a GTPase-activating protein (GAP) with activity towards its ARF domain. When overexpressed, ARD1 was associated with lysosomes and the Golgi apparatus. In agreement with this finding, lysosomal and Golgi membranes isolated from human liver by immunoaffinity contained native ARD1. ARD1, expressed as a green fluorescent fusion protein, was initially associated with the Golgi network and subsequently appeared on lysosomes, suggesting that ARD1 might undergo vectorial transport between the two organelles. Here we show by microscopic colocalization that GAP and ARF domains determine lysosomal and Golgi localization, respectively, consistent with the presence of more than one signal motif. Using truncated ARD1 molecules, expressed as green fluorescent fusion proteins, it was found that the signal for lysosomal localization was present in residues 301 to 402 of the GAP domain. Site-specific mutagenesis demonstrated that the sequence 369KXXXQ373 in the GAP domain was responsible for lysosomal localization. Association of ARD1 with the Golgi apparatus required tyrosine-based motifs. A green fluorescent fusion protein containing the QKQQQQF motif was partially associated with lysosomes, suggesting that this motif contains the information sufficient for lysosomal targeting. These results suggest that ARD1 is a multidomain protein with ARF and GAP regions, which contain Golgi and lysosomal localization signals, respectively, that could function in vesicular trafficking.

Vitale, Nicolas; Ferrans, Victor J.; Moss, Joel; Vaughan, Martha

2000-01-01

326

AChBP-targeted ?-conotoxin correlates distinct binding orientations with nAChR subtype selectivity  

PubMed Central

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel ?-conotoxin (?-TxIA) in the venom of Conus textile. ?-TxIA bound with high affinity to AChBPs from different species and selectively targeted the ?3?2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.

Dutertre, Sebastien; Ulens, Chris; Buttner, Regina; Fish, Alexander; van Elk, Rene; Kendel, Yvonne; Hopping, Gene; Alewood, Paul F; Schroeder, Christina; Nicke, Annette; Smit, August B; Sixma, Titia K; Lewis, Richard J

2007-01-01

327

Lysosomal Catabolism of a Protein Toxin: Staphylococcal Enterotoxin B.  

National Technical Information Service (NTIS)

Lysosomal proteases of rabbit liver, kidney and polymorphonuclear cells were tested for their ability to hydrolyze staphylococcal enterotoxin B, a protein exotoxin of Staphylococcus aureus. All lysosomal preparations effectively inactivated and catabolize...

P. G. Canonico

1973-01-01

328

Enhancement in selective mitochondrial association by direct modification of a mitochondrial targeting signal peptide on a liposomal based nanocarrier.  

PubMed

The focus of this study was on the development of a nano carrier targeted to mitochondria, a promising therapeutic drug target. We synthesized a lipid derivative that is conjugated with a mitochondrial targeting signal peptide (MTS), which permits the selective delivery of certain types of proteins to mitochondria. We then explored the use of an innovative technology in which MTS and the MITO-Porter were integrated. The latter is a liposome that delivers cargos to mitochondria via membrane fusion. The results indicate that the combination of MTS and the MITO-Porter would be useful for selective mitochondrial delivery via membrane fusion. PMID:23000575

Yamada, Yuma; Harashima, Hideyoshi

2012-09-19

329

Gordon Research Conference on Lysosomes Held in Andover, New Hampshire on July 3-8, 1994.  

National Technical Information Service (NTIS)

The Gordon Research Conference on Lysosomes was held at the Proctor Academy, Andover, New Hampshire on July 3-8, 1994. There were 146 participants at the meeting selected from over 200 applicants. The program included leaders from the U.S. and abroad in t...

A. Cruickshank

1994-01-01

330

A RANKL-PKC?-TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts  

PubMed Central

Bone resorption by osteoclasts requires a large number of lysosomes that release proteases in the resorption lacuna. Whether lysosomal biogenesis is a consequence of the action of transcriptional regulators of osteoclast differentiation or is under the control of a different and specific transcriptional pathway remains unknown. We show here, through cell-based assays and cell-specific gene deletion experiments in mice, that the osteoclast differentiation factor RANKL promotes lysosomal biogenesis once osteoclasts are differentiated through the selective activation of TFEB, a member of the MITF/TFE family of transcription factors. This occurs following PKC? phosphorylation of TFEB on three serine residues located in its last 15 amino acids. This post-translational modification stabilizes and increases the activity of this transcription factor. Supporting these biochemical observations, mice lacking in osteoclasts—either TFEB or PKC?—show decreased lysosomal gene expression and increased bone mass. Altogether, these results uncover a RANKL-dependent signaling pathway taking place in differentiated osteoclasts and culminating in the activation of TFEB to enhance lysosomal biogenesis—a necessary step for proper bone resorption.

Ferron, Mathieu; Settembre, Carmine; Shimazu, Junko; Lacombe, Julie; Kato, Shigeaki; Rawlings, David J.; Ballabio, Andrea; Karsenty, Gerard

2013-01-01

331

A RANKL-PKC?-TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts.  

PubMed

Bone resorption by osteoclasts requires a large number of lysosomes that release proteases in the resorption lacuna. Whether lysosomal biogenesis is a consequence of the action of transcriptional regulators of osteoclast differentiation or is under the control of a different and specific transcriptional pathway remains unknown. We show here, through cell-based assays and cell-specific gene deletion experiments in mice, that the osteoclast differentiation factor RANKL promotes lysosomal biogenesis once osteoclasts are differentiated through the selective activation of TFEB, a member of the MITF/TFE family of transcription factors. This occurs following PKC? phosphorylation of TFEB on three serine residues located in its last 15 amino acids. This post-translational modification stabilizes and increases the activity of this transcription factor. Supporting these biochemical observations, mice lacking in osteoclasts--either TFEB or PKC?--show decreased lysosomal gene expression and increased bone mass. Altogether, these results uncover a RANKL-dependent signaling pathway taking place in differentiated osteoclasts and culminating in the activation of TFEB to enhance lysosomal biogenesis-a necessary step for proper bone resorption. PMID:23599343

Ferron, Mathieu; Settembre, Carmine; Shimazu, Junko; Lacombe, Julie; Kato, Shigeaki; Rawlings, David J; Ballabio, Andrea; Karsenty, Gerard

2013-04-18

332

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

PubMed

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

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

2013-09-30

333

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

PubMed

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

McGaugh, S E; Janzen, F J

2011-01-24

334

Differential actions of insecticides on target sites: basis for selective toxicity  

PubMed Central

Whereas the selective toxicity of insecticides between insects and mammals has a long history of studies, it is now becoming abundantly clear that, in many cases, the differential action of insecticides on insects and mammalian target receptor sites is an important factor. In this paper, we first introduce the mechanism of action and the selective toxicity of pyrethroids as a prototype of study. Then, a more detailed account is given for fipronil, based primarily on our recent studies. Pyrethroids keep the sodium channels open for a prolonged period of time, causing elevation of the depolarizing after-potential. Once the after-potential reaches the threshold for excitation, repetitive after-discharges are produced, resulting in hyperexcitation of intoxicated animals. Only about 1% of sodium channels needs to be modified to produce hyperexcitation, indicating a high degree of toxicity amplification from sodium channels to animals. Pyrethroids were > 1000-fold more potent on cockroach sodium channels than rat sodium channels, and this forms the most significant factor to explain the selective toxicity of pyrethroids in insects over mammals. Fipronil, a phenylpyrazole, is known to act on the ?-aminobutyric acid receptor to block the chloride channel. It is effective against certain species of insects that have become resistant to most insecticides, including those acting on the ?-aminobutyric acid receptor, and is much more toxic to insects than to mammals. Recently, fipronil has been found to block glutamate-activated chloride channels in cockroach neurons in a potent manner. Since mammals are devoid of this type of chloride channel, fipronil block of the glutamate-activated chloride channel is deemed responsible, at least partially, for the higher selective toxicity to insects over mammals and for the lack of cross-resistance.

Narahashi, T; Zhao, X; Ikeda, T; Nagata, K; Yeh, JZ

2009-01-01

335

Manipulating Antigenic Ligand Strength to Selectively Target Myelin-Reactive CD4+ T Cells in EAE  

PubMed Central

The development of antigen-specific therapies for the selective tolerization of autoreactive T cells remains the Holy Grail for the treatment of T-cell-mediated autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This quest remains elusive, however, as the numerous antigen-specific strategies targeting myelin-specific T cells over the years have failed to result in clinical success. In this review, we revisit the antigen-based therapies used in the treatment of myelin-specific CD4+ T cells in the context of the functional avidity and the strength of signal of the encephalitogenic CD4+ T cell repertoire. In light of differences in activation thresholds, we propose that autoreactive T cells are not all equal, and therefore tolerance induction strategies must incorporate ligand strength in order to be successful in treating EAE and ultimately the human disease MS.

Sabatino, Joseph J.; Rosenthal, Kristen M.

2010-01-01

336

Manipulating antigenic ligand strength to selectively target myelin-reactive CD4+ T cells in EAE.  

PubMed

The development of antigen-specific therapies for the selective tolerization of autoreactive T cells remains the Holy Grail for the treatment of T-cell-mediated autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This quest remains elusive, however, as the numerous antigen-specific strategies targeting myelin-specific T cells over the years have failed to result in clinical success. In this review, we revisit the antigen-based therapies used in the treatment of myelin-specific CD4+ T cells in the context of the functional avidity and the strength of signal of the encephalitogenic CD4+ T cell repertoire. In light of differences in activation thresholds, we propose that autoreactive T cells are not all equal, and therefore tolerance induction strategies must incorporate ligand strength in order to be successful in treating EAE and ultimately the human disease MS. PMID:19904613

Sabatino, Joseph J; Rosenthal, Kristen M; Evavold, Brian D

2009-11-11

337

Deontic reasoning as a target of selection: Reply to Astington and Dack.  

PubMed

In their discussion of young children's deontic reasoning performance, Astington and Dack (2013) made the following claims: (1) Children need more cues to elicit cogent social norm reasoning than adults require, namely, explicit reference to authority; (2) Deontic reasoning improves with age, and this is evidence against a nativist view; (3) All evolutionary explanations of deontic reasoning advantages require positing a ''domain-specific deontic reasoning module."; and (4) young children excel at deontic reasoning because it is easier. Here, I refute each claim. Instead, I argue that (1) Social norm reasoning is one type of deontic reasoning that has been the target of selective pressure; (2) Development does not preclude nativism; (3) Epistemic utterances make no greater processing demands than deontic utterances; and (4) both adult and child norm reasoning performance is strongly influenced by reference to or implication of authority. PMID:23768759

Cummins, Denise Dellarosa

2013-06-14

338

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

PubMed

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

Luthra, Pratibha Mehta; Kumar, J B Senthil

2012-12-01

339

Cytochemical and biochemical identification of lysosomes in cryptococcus neoformans  

Microsoft Academic Search

Normaski optics, fluorescence and electron microscopy were employed to demonstrate the occurrence of lysosomes in capsulated, enzymatically decapsulated, and dewalled cells of a human isolate ofCryptococcus neoformans. Fluorescent studies, using acridine orange as a lysosomal indicator, revealed the presence of variously sized, spherical, reddish-orange fluorescing bodies. Electron microscopy studies demonstrated the presence of acid phosphatase (AP), a lysosome marker enzyme,

David L. Mason; Charles L. Wilson

1979-01-01

340

Gene Transfer Strategies for Correction of Lysosomal Storage Disorders  

Microsoft Academic Search

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

Alessandra d’Azzo

2003-01-01

341

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

PubMed

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

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

2013-04-15

342

Protein interactions: mapping interactome networks to support drug target discovery and selection.  

PubMed

Proteins are biomolecular structures that build the microscopic working machinery of any living system. Proteins within the cells and biological systems do not act alone, but rather team up into macromolecular structures enclosing intricate physicochemical dynamic connections to undertake biological functions. A critical step towards unraveling the complex molecular relationships in living systems is the mapping of protein-to-protein physical "interactions". The complete map of protein interactions that can occur in a living organism is called the "interactome". Achieving an adequate atlas of all the protein interactions within a living system should allow to build its interaction network and to identity the "central nodes" that can be critical for the function, the homeostasis, and the movement of such system. Focusing on human studies, the data about the human interactome are most relevant for current biomedical research, because it is clear that the location of the proteins in the interactome network will allow to evaluate their centrality and to redefine the potential value of each protein as a drug target. This chapter presents our current knowledge on the human protein-protein interactome and explains how such knowledge can help us to select adequate targets for drugs. PMID:22821600

De Las Rivas, Javier; Prieto, Carlos

2012-01-01

343

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

PubMed Central

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

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

2010-01-01

344

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

PubMed

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

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

2011-11-23

345

Lysosomal storage diseases-the horizon expands.  

PubMed

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

Boustany, Rose-Mary Naaman

2013-08-13

346

Lysosomal Activity Associated with Developmental Axon Pruning  

Microsoft Academic Search

Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, which generates considerable cellular debris, is synapse elimination, in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of

Jae W. Song; Thomas Misgeld; Hyuno Kang; Sharm Knecht; Ju Lu; Yi Cao; Susan L. Cotman; Derron L. Bishop; Jeff W. Lichtman

2008-01-01

347

Update on treatment of lysosomal storage diseases  

PubMed Central

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

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

2007-01-01

348

Modifications of lysosomal enzymes in Dictyostelium discoideum  

Microsoft Academic Search

This paper has two purposes. The first is to review the past studies on the structure, biosynthesis, and immunological properties of a class of glycoproteins, the lysosomal enzymes, in Dictyostelium discoideum. The second purpose is to present new data on the analysis of mutant strains altered in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides, and on the characterization

Hudson H. Freeze

1986-01-01

349

Prolylcarboxypeptidase: A Recently Described Lysosomal Enzyme.  

National Technical Information Service (NTIS)

Prolylcarboxypeptidase (PCP) was first found in the kidney. In addition to the kidney, PCP occurs in human plasma or erythrocytes. PCP occurs in a lysosomal fraction of swine kidney cortex and of human granulocytes. Table III shows the distribution of PCP...

H. Y. T. Yang E. G. Erdos

1971-01-01

350

Vincristine Induces Dramatic Lysosomal Changes and Sensitizes Cancer Cells to Lysosome-Destabilizing Siramesine  

Microsoft Academic Search

Vincristine is a microtubule-destabilizing antimitotic drug that has been used in cancer therapy for over 40 years. However, the knowledge on vincristine-induced cell death pathways is still sparse. Here, we show that vincristine induces dramatic changes in the lysosomal compartment and sensi- tizes cells to lysosomal membrane permeabilization. In HeLa cervix carcinoma cells, vincristine induced mitotic arrest and massive cell

Line Groth-Pedersen; Marie Stampe Ostenfeld; Maria Høyer-Hansen; Jesper Nylandsted; Marja Jaattela

2007-01-01

351

Antihypertensive effects of selective prostaglandin E2 receptor subtype 1 targeting  

PubMed Central

Clinical use of prostaglandin synthase–inhibiting NSAIDs is associated with the development of hypertension; however, the cardiovascular effects of antagonists for individual prostaglandin receptors remain uncharacterized. The present studies were aimed at elucidating the role of prostaglandin E2 (PGE2) E-prostanoid receptor subtype 1 (EP1) in regulating blood pressure. Oral administration of the EP1 receptor antagonist SC51322 reduced blood pressure in spontaneously hypertensive rats. To define whether this antihypertensive effect was caused by EP1 receptor inhibition, an EP1-null mouse was generated using a “hit-and-run” strategy that disrupted the gene encoding EP1 but spared expression of protein kinase N (PKN) encoded at the EP1 locus on the antiparallel DNA strand. Selective genetic disruption of the EP1 receptor blunted the acute pressor response to Ang II and reduced chronic Ang II–driven hypertension. SC51322 blunted the constricting effect of Ang II on in vitro–perfused preglomerular renal arterioles and mesenteric arteriolar rings. Similarly, the pressor response to EP1-selective agonists sulprostone and 17-phenyltrinor PGE2 were blunted by SC51322 and in EP1-null mice. These data support the possibility of targeting the EP1 receptor for antihypertensive therapy.

Guan, Youfei; Zhang, Yahua; Wu, Jing; Qi, Zhonghua; Yang, Guangrui; Dou, Dou; Gao, Yuansheng; Chen, Lihong; Zhang, Xiaoyan; Davis, Linda S.; Wei, Mingfeng; Fan, Xuefeng; Carmosino, Monica; Hao, Chuanming; Imig, John D.; Breyer, Richard M.; Breyer, Matthew D.

2007-01-01

352

Tumorigenic Polyploid Cells Contain Elevated ROS and are Selectively Targeted by Antioxidant Treatment  

PubMed Central

Polyploidy has been linked to tumorigenicity mainly due to the chromosomal aberrations. Elevated reactive oxygen species (ROS) generation, on the other hand, has also been associated with oncogenic transformation in most cancer cells. However, a possible link between ploidy and ROS is largely unexplored. Here we have exemined the role of ROS in the tumorigenicity of polyploid cells. We show that polyploid prostate and mammary epithelial cells contain higher levels of ROS due to their higher mitochondrial contents. ROS levels and mitochondrial mass are also higher in dihydrocytochalasin B (DCB)-induced polyploid cells, suggesting that higher levels of ROS observed in polyploid cell can occur due to cytokinesis failure. Interestingly, polyploid cells were more sensitive to the inhibitory effect of the antioxidant, N-Acetyl-L-cysteine (NAC), than control diploid cells. Treatment of polyploid/diploid cells with NAC led to the selective elimination of polyploid cells over time and abrogated the tumorigenicity of polyploid cells. This effect was partially mediated via the Akt signaling pathway. We next explored a possible role for ROS in promoting chromosomal instability by analyzing the effects of ROS on the mitotic stage of the cell cycle. Enhancing ROS levels by treating cells with hydrogen peroxide delayed not only entry into and but also exit from mitosis. Furthermore, increasing ROS levels significantly increased taxol resistance. Our results indicated that increased ROS in polyploid cells can contribute to tumorigenicity and highlight the therapeutic potential of antioxidants by selectively targeting the tumorigenic polyploid cells and by reversing taxol resistance.

Roh, Meejeon; van der Meer, Riet; Abdulkadir, Sarki A.

2011-01-01

353

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

PubMed

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 global 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 Man(9)GlcNAc(2) 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 eight glycans from 250 cancer cells and five 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

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

2012-07-23

354

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

Microsoft Academic Search

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

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

2000-01-01

355

Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process  

PubMed Central

In this study, we report a novel mechanism of action for a cytotoxic derivative of betulinic acid (BA). B10 is a semi-synthetic glycosylated derivative of BA selected for its enhanced cytotoxic activity. Interestingly, although B10 induces apoptosis, caspase-3 downregulation incompletely prevents B10-induced cell death, Bcl-2 overexpression fails to protect cells and DNA fragmentation rates do not reflect cell death rates in contrast to cytoplasmic membrane permeabilization. These results implicate that apoptotic and non-apoptotic cell death coexist upon B10 treatment. Unexpectedly, we found that B10 induces autophagy and also abrogates the autophagic flux. B10 destabilizes lysosomes as shown by Lysotracker Red staining and by cathepsin Z and B release from lysosomes into the cytoplasm. Consistently, the cathepsin inhibitor Ca074Me significantly decreases B10-induced cell death, further supporting the fact that the release of lysosomal enzymes contributes to B10-triggered cell death. Downregulation of ATG7, ATG5 or BECN1 by RNAi significantly decreases caspase-3 activation, lysosomal permeabilization and cell death. Thus, by concomitant induction of autophagy and inhibition of the autophagic flux, B10 turns autophagy into a cell death mechanism. These findings have important implications for the therapeutic exploitation of BA derivatives, particularly in apoptosis-resistant cancers.

Gonzalez, P; Mader, I; Tchoghandjian, A; Enzenmuller, S; Cristofanon, S; Basit, F; Debatin, K-M; Fulda, S

2012-01-01

356

Chondroitin sulfate is involved in lysosomal transport of lysozyme in U937 cells.  

PubMed

Human promonocytes U937 synthesize lysozyme and retain approximately one third of it within lysosomes. Lysozyme is not glycosylated; thus, it cannot be subject to mannose-6-phosphate-dependent targeting to lysosomes. It is a basic protein with a pI of 10.5 and is known to interact with negatively charged macromolecules like proteoglycans. Therefore, we examined whether the latter are involved in the lysosomal targeting of lysozyme in U937 cells. We partially diminished the electronegative charge of newly synthesized proteoglycans by inhibiting their sulfation with chlorate. This increased the rate of secretion of lysozyme. Upon treatment of U937 cells with phorbol esters, the rate of secretion of lysozyme was increased to more than 90%. This coincided with an almost complete redistribution of a [(35)S]sulfate bearing proteoglycan to the secretory pathway. After a brief pulse with [(35)S]sulfate in the control, 80% of the [(35)S]sulfate-bearing proteoglycan was retained within the cells, whereas in the treated cells this proportion was decreased to 13%. The secreted proteoglycan was sensitive to chondroitinase ABC and bound to immobilized lysozyme. This interaction was disrupted by 50-300 mM NaCl. The intracellularly retained proteoglycan was degraded with a half-life of 50-60 minutes and seemed to be directed to lysosomes because in the presence of NH(4)Cl the degradation was strongly inhibited. Our results suggest that the proteoglycan is involved in lysosomal targeting of lysozyme in U937 cells. PMID:11148136

Lemansky, P; Hasilik, A

2001-01-01

357

Monitoring lysosomal activity in nanoparticle-treated cells.  

PubMed

Certain nanoparticles have been shown to accumulate within lysosome and hence may cause lysosomal pathologies such as phospholipidosis, lysosomal overload, and autophagy. This chapter describes a method for evaluation of lysosomal activity in porcine kidney cells (LLC-PK1) after exposure to nanoparticles. This method uses the accumulation of a cationic fluorescent dye (LysoTracker Red) in acidic cellular compartments as an indicator of total lysosome content. The lysotracker signal is normalized to the signal from a thiol-reactive dye which is proportional to the total number of viable cells. PMID:21116970

Neun, Barry W; Stern, Stephan T

2011-01-01

358

Siramesine triggers cell death through destabilisation of mitochondria, but not lysosomes.  

PubMed

A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20??M. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20??M siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5-40??M); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant ?-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15??M, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine analogues with high anticancer potential. PMID:24091661

Hafner ?esen, M; Repnik, U; Turk, V; Turk, B

2013-10-03

359

ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage.  

PubMed

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD(50) light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage. PMID:22889762

Kessel, David H; Price, Michael; Reiners, John J

2012-08-14

360

ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage  

PubMed Central

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD50 light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.

Kessel, David H.; Price, Michael; Reiners, Jr., John J.

2012-01-01

361

Reactivity of lysosomes to inside-out cell membrane vesicles in a cell-free system.  

PubMed

To study ultrastructurally the mechanisms of lysosome reactions to cell membrane-derived intracellular membranes we developed a cell free system using small inside-out and rightside-out cell membrane vesicles (IOVs and ROVs) as a target of the reactions. The IOVs were generated from rat erythrocyte ghosts in a low ionic strength alkaline solution in the absence of divalent cations after erythrocytes were reacted with wheat germ agglutinin-coated colloidal gold [WGA (CG)], while ROVs were from ghosts homogenized in a buffer with MgSO4 and bovine serum albumin-coated CG [BSA (CG)]. WGA (CG)s bound to the cell surface were rearranged on the membrane and distributed irregularly on the inner surface of generated small IOVs. A coat structure derived from the ghost's submembranous coat was almost depleted from their outer surface. By contrast, BSA (CG)-binding to the membranes was negligible in the process of ROV formation. When isolated rat liver lysosomes were incubated with these WGA (CG)-binding small IOVs at 37 degrees C, CG particles were found in several lysosomes under electron microscopy. Some lysosomes adhered to the IOVs, and their limiting membranes were found to collapse and disappear partially at the adhering region, suggesting their fusion. This reaction seems to occur even in cytosol-free solution. By contrast, the lysosomes indicated very low reaction to BSA (CG)-containing ROV, and to WGA (CG) or BSA (CG) alone. Therefore, it is suggested that isolated liver lysosomes react, at least to fuse, in a cytosol-independent fashion, with surface coat-depleted IOVs derived from WGA (CG)-bound and then -rearranged erythrocyte membranes. PMID:8069943

Kawai, N; Ichihara, I

1994-02-01

362

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

PubMed Central

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).

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

2012-01-01

363

Dynamics of saccade target selection: Race model analysis of double step and search step saccade production in human and macaque  

PubMed Central

We investigated how saccade target selection by humans and macaque monkeys reacts to unexpected changes of the image. This was explored using double step and search step tasks in which a target, presented alone or as a singleton in a visual search array, steps to a different location on infrequent, random trials. We report that human and macaque monkey performance are qualitatively indistinguishable. Performance is stochastic with the probability of producing a compensated saccade to the final target location decreasing with the delay of the step. Compensated saccades to the final target location are produced with latencies relative to the step that are comparable to or less than the average latency of saccades on trials with no target step. Noncompensated errors to the initial target location are produced with latencies less than the average latency of saccades on trials with no target step. Noncompensated saccades to the initial target location are followed by corrective saccades to the final target location following an intersaccade interval that decreases with the interval between the target step and the initiation of the noncompensated saccade. We show that this pattern of results cannot be accounted for by a race between two stochastically independent processes producing the saccade to the initial target location and another process producing the saccade to the final target location. However, performance can be accounted for by a race between three stochastically independent processes – a GO process producing the saccade to the initial target location, a STOP process interrupting that GO process, and another GO process producing the saccade to the final target location. Furthermore, if the STOP process and second GO process start at the same time, then the model can account for the incidence and latency of mid-flight corrections and rapid corrective saccades. This model provides a computational account of saccade production when the image changes unexpectedly.

Camalier, C. R.; Gotler, A.; Murthy, A.; Thompson, K.G.; Logan, G.D.; Palmeri, T.J.; Schall, J.D.

2007-01-01

364

hVps41 and VAMP7 function in direct TGN to late endosome transport of lysosomal membrane proteins.  

PubMed

Targeted delivery of lysosome-associated membrane proteins is important for lysosome stability and function. Here we identify a pathway for transport of lysosome-associated membrane proteins directly from the trans-Golgi network to late endosomes, which exists in parallel to mannose 6-phosphate receptor and clathrin-dependent transport of lysosomal enzymes to early endosomes. By immunoelectron microscopy we localized endogenous LAMP-1 and -2 as well as LAMP-1-mGFP to non-coated, biosynthetic carriers at the trans-Golgi network and near late endosomes. These LAMP carriers were negative for mannose 6-phosphate receptor, adaptor-protein complex-1, secretory albumin and endocytic markers, but contained the homotypic fusion and protein sorting complex component hVps41 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein VAMP7. Knockdown of hVps41 or VAMP7 resulted in the accumulation of lysosome-associated membrane protein carriers, whereas knockdown of hVps39 or hVps18 did not, indicating that the effect of hVps41 is independent of CORVET/HOPS. Mannose 6-phosphate receptor carriers remained unaffected upon hVps41 or VAMP7 knockdown, implicating that hVps41 and VAMP7 are specifically involved in the fusion of trans-Golgi network-derived lysosome-associated membrane protein carriers with late endosomes. PMID:23322049

Pols, Maaike S; van Meel, Eline; Oorschot, Viola; ten Brink, Corlinda; Fukuda, Minoru; Swetha, M G; Mayor, Satyajit; Klumperman, Judith

2013-01-01

365

Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus  

NASA Astrophysics Data System (ADS)

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.

Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

2012-10-01

366

Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus  

PubMed Central

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.

Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

2012-01-01

367

Molecular pathologies of and enzyme replacement therapies for lysosomal diseases.  

PubMed

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

Sakuraba, Hitoshi; Sawada, Makoto; Matsuzawa, Fumiko; Aikawa, Sei-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Itoh, Kohji

2006-08-01

368

Neural Control of Visual Search by Frontal Eye Field: Effects of Unexpected Target Displacement on Visual Selection and Saccade Preparation  

PubMed Central

The dynamics of visual selection and saccade preparation by the frontal eye field was investigated in macaque monkeys performing a search-step task combining the classic double-step saccade task with visual search. Reward was earned for producing a saccade to a color singleton. On random trials the target and one distractor swapped locations before the saccade and monkeys were rewarded for shifting gaze to the new singleton location. A race model accounts for the probabilities and latencies of saccades to the initial and final singleton locations and provides a measure of the duration of a covert compensation process—target-step reaction time. When the target stepped out of a movement field, noncompensated saccades to the original location were produced when movement-related activity grew rapidly to a threshold. Compensated saccades to the final location were produced when the growth of the original movement-related activity was interrupted within target-step reaction time and was replaced by activation of other neurons producing the compensated saccade. When the target stepped into a receptive field, visual neurons selected the new target location regardless of the monkeys’ response. When the target stepped out of a receptive field most visual neurons maintained the representation of the original target location, but a minority of visual neurons showed reduced activity. Chronometric analyses of the neural responses to the target step revealed that the modulation of visually responsive neurons and movement-related neurons occurred early enough to shift attention and saccade preparation from the old to the new target location. These findings indicate that visual activity in the frontal eye field signals the location of targets for orienting, whereas movement-related activity instantiates saccade preparation.

Murthy, Aditya; Ray, Supriya; Shorter, Stephanie M.; Schall, Jeffrey D.; Thompson, Kirk G.

2009-01-01

369

Wave-number selection by target patterns and sidewalls in Rayleigh-Bénard convection  

NASA Astrophysics Data System (ADS)

We present experimental results for patterns of Rayleigh-Bénard convection in a cylindrical container with static sidewall forcing. The fluid used was methanol, with a Prandlt number ?=7.17 , and the aspect ratio was ??R/d?19 ( R is the radius and d the thickness of the fluid layer). In the presence of a small heat input along the sidewall, a sudden jump of the temperature difference ?T from below to slightly above a critical value ?Tc produced a stable pattern of concentric rolls (a target pattern) with the central roll (the umbilicus) at the center of the cell. A quasistatic increase of ???T/?Tc-1 beyond ?1,c?0.8 caused the umbilicus of the pattern to move off center. As observed by others, a further quasistatic increase of ? up to ?=15.6 caused a sequence of transitions at ?i,b,i=1,...,8 , each associated with the loss of one convection roll at the umbilicus. Each loss of a roll was preceded by the displacement of the umbilicus away from the center of the cell. After each transition the umbilicus moved back toward but never quite reached the center. With decreasing ? new rolls formed at the umbilicus when ? was reduced below ?i,aselected in the far field of an infinitely extended target pattern. To our knowledge there is as yet no prediction for the wave number selected by the umbilicus itself, or by the cell wall of the finite experimental system.

Royer, John R.; O'Neill, Patrick; Becker, Nathan; Ahlers, Guenter

2004-09-01

370

Emerging drug discovery approaches for selective targeting of "precursor" metastatic breast cancer cells: highlights and perspectives  

PubMed Central

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

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

2011-01-01

371

Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.  

PubMed

In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced a caspase-dependent apoptotic response, being activation of caspase-8, -9 and -3 the result of a post-mitochondrial event. PMID:23994488

Marino, Julieta; García Vior, María C; Furmento, Verónica A; Blank, Viviana C; Awruch, Josefina; Roguin, Leonor P

2013-08-28

372

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

PubMed Central

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

2012-01-01

373

Engineering nanostructures with enhanced thermoplasmonic properties for biosensing and selective targeting applications.  

PubMed

This paper connects the study of thermoplasmonic properties in nanoscale particles with areas of biophysics involving a cell membrane with or without conductive pores. Using a quasistatic finite element modeling of the heat transfer equation in three dimensions we simulate the stationary heat generation and temperature field around several types of gold-based nanostructures. Models were constructed that emphasized the importance of obtaining precise temperature fields that might subsequently be used for biosensing and selective targeting applications. By analyzing the observed temperature increase, effective complex permittivity, and electric field enhancement that result from plasmonic resonance, this theoretical framework provides insight into the role of the nanoparticle shape in heat generation. To illustrate the usefulness of this approach for biosensing applications, we consider how the positioning of the nanoantenna affects heating efficiency. Linear response calculations of the temperature increase reveal that symmetric gold nanosphere dimers are not only suitable for sensing applications, but can also play the role of heat sources which are more efficient than the case of a single nanosphere. We also predict that this specific type of nanoantenna allows us to detect the presence and size of a hole in the cell membrane. These results provide insight into the physics of the cell membrane and provide guidance for more detailed studies of the nanoscale control of temperature in biological materials. PMID:23410374

Essone Mezeme, M; Brosseau, C

2013-01-28

374

Estrogen receptor ? can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation  

PubMed Central

Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ER?) receptors. More specifically, ER? represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ER? represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ER? can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells.

Marques, Maud; Laflamme, Liette; Gaudreau, Luc

2013-01-01

375

Rh(DIP)3(3+): a shape-selective metal complex which targets cruciforms.  

PubMed Central

The coordination complex tris(4,7-diphenylphenanthroline)rhodium(III), Rh(DIP)3(3+), binds to and, upon photoactivation, cleaves both DNA strands near the base of a DNA cruciform. Sites of photoinduced double-stranded DNA cleavage by the rhodium complex map to regions containing cruciforms on closed circular pBR322, pColE1 and phi X174 (replicative form) DNAs. Neither cleavage nor binding by the metal complex, assayed using S1 nuclease, is found on the linear plasmid which lacks the extruded cruciform. High resolution mapping experiments reveal that Rh(DIP)3(3+) cleaves at a specific AT-rich site neighboring the stem of the minor cruciform on pBR322. The primary site of cleavage is found at position 3238 on the 3'-strand and 3250 on the 5'-strand and is remarkably specific. The pattern of cleavage, to one side only of the cruciform stem, indicates an asymmetry in the cruciform structure recognized by the complex. These results suggest that Rh(DIP)3(3+) may provide a useful reagent to probe cruciform sites. In addition, the high degree of specificity found in targeting the cruciform structure with this simple metal complex underscores the utility of shape-selection for the recognition of specific sites on a DNA strand. Images

Kirshenbaum, M R; Tribolet, R; Barton, J K

1988-01-01

376

Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs  

PubMed Central

Background 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology/Principal Findings Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. Conclusion/Significance 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.

Tribouillard-Tanvier, Deborah; Dos Reis, Suzana; Gug, Fabienne; Voisset, Cecile; Beringue, Vincent; Sabate, Raimon; Kikovska, Ema; Talarek, Nicolas; Bach, Stephane; Huang, Chenhui; Desban, Nathalie; Saupe, Sven J.; Supattapone, Surachai; Thuret, Jean-Yves; Chedin, Stephane; Vilette, Didier; Galons, Herve; Sanyal, Suparna; Blondel, Marc

2008-01-01

377

Screening method using selected reaction monitoring for targeted proteomics studies of nasal lavage fluid.  

PubMed

Proteomic-based studies of nasal lavage fluid (NLF) may identify molecular pathways associated with disease pathology and new biomarker candidates of upper airway diseases. However, most studies have used rather tedious untargeted MS techniques. Selected reaction monitoring (SRM) is a sensitive and specific technique that can be used with high throughput. In this study, we developed a semiquantitative SRM-based method targeting 244 NLF proteins. The protein set was identified through a literature study in combination with untargeted LC-MS/MS analyses of trypsin-digested NLF samples. The SRM assays were designed using MS/MS data either downloaded from a proteomic data repository or experimentally obtained. Each protein is represented by one to five peptides, resulting in 708 SRM assays. Three to four transitions per assay were used to ensure analyte specificity. The majority (69%) of the assays showed good within-day precision (coefficient of variation ? 20%). The accuracy of the method was evaluated by analyzing four samples prepared with varying amounts of four proteins. Peptide and protein ratios were in good agreement with expected ratios. In conclusion, a high throughput screening method for relative quantification of 244 NLF proteins was developed. The method should be of general use in any proteomic study of the upper airways. PMID:23214469

Mörtstedt, Harriet; Kåredal, Monica H; Jönsson, Bo A G; Lindh, Christian H

2012-12-14

378

Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes.  

PubMed

A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. Here we describe a new negative regulator of JAK/STAT signaling and identify a potential mechanism by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, we have identified Ken & Barbie (Ken) , which is an ortholog of the mammalian proto-oncogene BCL6 , as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, we show that endogenous Ken specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo. PMID:16401426

Arbouzova, Natalia I; Bach, Erika A; Zeidler, Martin P

2006-01-10

379

Lysosomal Degradation of Ubiquitin-Tagged Receptors  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. Cytosolic proteins are tagged with the polypeptide ubiquitin for eventual destruction by the proteasome. A recent paper in Cell (L. Hicke and H. Riezman, vol. 84, p. 277) shows that in yeast ubiquitin also serves to tag membrane proteins for degradation by proteases in the vacuole, the yeast equivalent of the lysosome.

Stella M. Hurtley (AAAS;Science Magazine, Europe Office)

1996-02-02

380

Therapeutic approaches for neuronopathic lysosomal storage disorders  

Microsoft Academic Search

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

Raphael Schiffmann

2010-01-01

381

Lysosomal Glycosphingolipid Recognition by NKT Cells  

Microsoft Academic Search

NKT cells represent a distinct lineage of T cells that coexpress a conserved alphabeta T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized

Dapeng Zhou; Jochen Mattner; Carlos Cantu; Nicolas Schrantz; Ning Yin; Ying Gao; Yuval Sagiv; Kelly Hudspeth; Yun-Ping Wu; Tadashi Yamashita; Susann Teneberg; Dacheng Wang; Richard L. Proia; Steven B. Levery; Paul B. Savage; Luc Teyton; Albert Bendelac

2004-01-01

382

Glycosylation and Phosphorylation of Lysosomal Glycosylasparaginase  

Microsoft Academic Search

Glycosylasparaginase (EC 3.5.1.26) is a lysosomal amidase which hydrolyzes the bond between asparagine and the sugar moiety in N-linked glycoproteins. Deficiency of the enzyme results in aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation. Mature enzyme is formed by two proteolytic cleavage steps subsequent to removal of its signal peptide: (1) an activation cleavage in the ER of the

Michelle Vettese-Dadey

1996-01-01

383

Genetic Counseling for Lysosomal Storage Diseases  

Microsoft Academic Search

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

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

384

Indium-111-diethylenetriaminepentaacetic acid-octreotide is delivered in vivo to pancreatic, tumor cell, renal, and hepatocyte lysosomes.  

PubMed

To better understand the factors that govern the target-to-background ratios of 111In-diethylenetriaminepentaacetic acid (DTPA) polypeptides, we studied 111In-DTPA-octreotide and a model nontargeted compound, 111In-DTPA-poly(D)lysine-biotin. We evaluated the fate of 111In-DTPA-octreotide after it localizes in somatostatin receptor-positive tissues and sought to determine why such a large fraction of these and other 111In-DTPA-polypeptides accumulate in the liver and kidneys. Biodistribution studies in rats with an implanted pancreatic adenocarcinoma demonstrated rapid accumulation of 111In-DTPA-octreotide in the pancreas and tumor. Indium-111 also accumulated in the liver and kidneys. Subcellular fractionation of the liver, kidneys, tumor, and pancreas showed that the majority of the radioactivity copurified with lysosomal enzymes. Even at 1 h, little radioactivity was found in the fractions containing a cell surface enzyme. This suggests that in each tissue, the 111In-DTPA-octreotide was rapidly shuttled from the cell surface to lysosomes. In the liver, hepatocyte lysosomes were separated from sinusoidal and Kupffer cell lysosomes by administering chloroquine prior to sacrifice. This density shift experiment indicated that 111In-DTPA-octreotide accumulated predominantly in hepatocyte lysosomes. A low molecular weight 111In-DTPA-poly(D)lysine-biotin compound was synthesized, and biodistribution studies showed substantial renal accumulation. The poly(D)lysine backbone conferred resistance to degradation, and this fact allowed determination of the distribution of this compound at the cellular level using an antibiotin antibody and immunohistochemical techniques. These experiments, as well as subcellular fractionation studies, demonstrated that the 111In-DTPA-poly(D)lysine-biotin compound accumulated in the lysosomes of proximal renal tubular cells. These results indicate that lysosomes play a critical role in the cellular physiology of radiolabeled polypeptides. Using these data, we propose a comprehensive model that summarizes the factors that govern the target to background ratios of radiolabeled polypeptides. PMID:9044843

Duncan, J R; Stephenson, M T; Wu, H P; Anderson, C J

1997-02-15

385

Inter-surgeon variability in the selection of anterior and posterior commissures and its potential effects on target localization  

PubMed Central

Background This study reports the inter-surgeon variability in manual selection of the anterior and posterior commissures (AC and PC). The study also investigates the effect of this variability on the localization of targets like STN, Vim and GPi. The additional effect of variation in the selection of the mid-plane on target localization is also evaluated. Methods 43 neurosurgeons (38 attendings, 5 residents/fellows) were asked to select the AC and the PC points (as routinely used for stereotactic neurosurgical planning) on two MRI scans. The corresponding mid-commissural points (MCP) and target coordinates were calculated. Results The collected data show that the MCP point is more reliable than either the AC or the PC points. This data also show that, even for experienced neurosurgeons, variations in selecting the AC and the PC point results in substantial variations at the target points: 1.15 ± 0.89mm, 1.45 ± 1.25mm, 1.21 ± 0.83 for the STN, Vim, and GPi, respectively for the first MR volume and 1.08 ± 1.37mm, 1.35 ± 1.71mm, 1.12 ± 1.17mm for the same structures for the second volume. These variations are larger when residents/fellows are included in the data set. Conclusions The data collected in this study highlights the difficulty of establishing a common reference system that can be used to communicate target location across sites. It indicates the need for the development and evaluation of alternative normalization methods that would permit specifying targets directly in image coordinates or the development of improved imaging techniques that would permit direct targeting.

Pallavaram, Srivatsan; Yu, Hong; Spooner, John; D'Haese, Pierre-Francois; Bodenheimer, Bobby; Konrad, Peter E; Dawant, Benoit M

2009-01-01

386

Effective and selective targeting of leukemia cells using a TORC1\\/2 kinase inhibitor  

Microsoft Academic Search

Targeting the mammalian target of rapamycin (mTOR) protein is a promising strategy for cancer therapy. The mTOR kinase functions in two complexes, TORC1 (target of rapamycin complex-1) and TORC2 (target of rapamycin complex-2); however, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. We compared rapamycin with PP242, an inhibitor of the active site

Matthew R Janes; Jose J Limon; Lomon So; Jing Chen; Raymond J Lim; Melissa A Chavez; Collin Vu; Michael B Lilly; Sharmila Mallya; S Tiong Ong; Marina Konopleva; Michael B Martin; Pingda Ren; Yi Liu; Christian Rommel; David A Fruman

2010-01-01

387

The Role of Microscopy in Understanding Atherosclerotic Lysosomal Lipid Metabolism  

NASA Astrophysics Data System (ADS)

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with mod